Expression vector encoding apoptosis-specific eIF-5A

ABSTRACT

The present invention relates to isolated and/or purified rat apoptosis-specific eucaryotic initiation Factor-5A (eIF-5A) and deoxyhypusine synthase (DHS) nucleic acids and polypeptides. The present invention also relates to methods of modulating apoptosis using apoptosis-specific eIF-5A and DHS, and antisense oligonucleotides and expression vectors of apoptosis-specific eIF-5A and DHS useful in such methods.

RELATED APPLICATIONS

This application claims priority to, and is a continuation of, U.S.application Ser. No. 10/141,647, filed on May 7, 2002 U.S. Pat. No.7,166,467, which is a continuation-in-part of U.S. Ser. No. 09/909,796,filed Jul. 23, 2001, now U.S. Pat. No. 6,867,237, issued Mar. 15, 2005,the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to apoptosis-specific eucaryoticinitiation Factor-5A (eIF-5A) and deoxyhypusine synthase (DHS) nucleicacids and polypeptides and methods for modulating apoptosis in cellsusing apoptosis-specific eIF-5A and DHS.

BACKGROUND OF THE INVENTION

Apoptosis is a genetically programmed cellular event that ischaracterized by well-defined morphological features, such as cellshrinkage, chromatin condensation, nuclear fragmentation, and membraneblebbing. Kerr et al. (1972) Br. J. Cancer, 26, 239-257; Wyllie et al.(1980) Int. Rev. Cytol., 68, 251-306. It plays an important role innormal tissue development and homeostasis, and defects in the apoptoticprogram are thought to contribute to a wide range of human disordersranging from neurodegenerative and autoimmunity disorders to neoplasms.Thompson (1995) Science, 267, 1456-1462; Mullauer et al. (2001) Mutat.Res, 488, 211-231. Although the morphological characteristics ofapoptotic cells are well characterized, the molecular pathways thatregulate this process have only begun to be elucidated.

One group of proteins that is thought to play a key role in apoptosis isa family of cysteine proteases, termed caspases, which appear to berequired for most pathways of apoptosis. Creagh & Martin (2001) Biochem.Soc. Trans, 29, 696-701; Dales et al. (2001) Leuk. Lymphoma, 41,247-253. Caspases trigger apoptosis in response to apoptotic stimuli bycleaving various cellular proteins, which results in classicmanifestations of apoptosis, including cell shrinkage, membrane blebbingand DNA fragmentation. Chang & Yang (2000) Microbiol. Mol. Biol. Rev.,64, 821-846.

Pro-apoptotic proteins, such as Bax or Bak, also play a key role in theapoptotic pathway by releasing caspase-activating molecules, such asmitochondrial cytochrome c, thereby promoting cell death throughapoptosis. Martinou & Green (2001) Nat. Rev. Mol. Cell. Biol., 2, 63-67;Zou et al. (1997) Cell, 90, 405-413. Anti-apoptotic proteins, such asBcl-2, promote cell survival by antagonizing the activity of thepro-apoptotic proteins, Bax and Bak. Tsujimoto (1998) Genes Cells, 3,697-707; Kroemer (1997) Nature Med., 3, 614-620. The ratio of Bax:Bcl-2is thought to be one way in which cell fate is determined; an excess ofBax promotes apoptosis and an excess of Bcl-2 promotes cell survival.Salomons et al. (1997) Int. J. Cancer, 71, 959-965; Wallace-Brodeur &Lowe (1999) Cell Mol. Life Sci.,55, 64-75.

Another key protein involved in apoptosis is that encoded by the tumorsuppressor gene p53. This protein is a transcription factor thatregulates cell growth and induces apoptosis in cells that are damagedand genetically unstable, presumably through upregulation of Bax. Boldet al. (1997) Surgical Oncology, 6, 133-142; Ronen et al., 1996; Schuler& Green (2001) Biochem. Soc. Trans., 29, 684-688; Ryan et al. (2001)Curr. Opin. Cell Biol., 13, 332-337; Zörnig et al. (2001) Biochem.Biophys. Acta, 1551, F1-F37.

The distinct morphological features that characterize cells undergoingapoptosis have given rise to a number of methods for assessing the onsetand progress of apoptosis. One such feature of apoptotic cells that canbe exploited for their detection is activation of a flippase, whichresults in externalization of phosphatidylserine, a phospholipidnormally localized to the inner leaflet of the plasma membrane. Fadok etal. (1992) J. Immunol., 149, 4029-4035. Apoptotic cells bearingexternalized phosphatidylserine can be detected by staining with aphosphatidylserine-binding protein, Annexin V, conjugated to afluorescent dye. The characteristic DNA fragmentation that occurs duringthe apoptotic process can be detected by labeling the exposed 3′-OH endsof the DNA fragments with fluorescein-labeled deoxynucleotides.Fluorescent dyes that bind nucleic acids, such as Hoerscht 33258, can beused to detect chromatin condensation and nuclear fragmentation inapoptotic cells. The degree of apoptosis in a cell population can alsobe inferred from the extent of caspase proteolytic activity present incellular extracts.

As a genetically defined process, apoptosis, like any otherdevelopmental program, can be disrupted by mutation. Alterations in theapoptotic pathways are believed to play a key role in a number ofdisease processes, including cancer. Wyllie et al. (1980) Int. Rev.Cytol., 68, 251-306; Thompson (1995) Science, 267, 1456-1462; Sen &D'Incalci (1992) FEBS Letters, 307, 122-127; McDonnell et al. (1995)Seminars in Cancer and Biology, 6, 53-60. Investigations into cancerdevelopment and progression have traditionally been focused on cellularproliferation. However, the important role that apoptosis plays intumorigenesis has recently become apparent. In fact, much of what is nowknown about apoptosis has been learned using tumor models, since thecontrol of apoptosis is invariably altered in some way in tumor cells.Bold et al. (1997) Surgical Oncology, 6, 133-142.

Apoptosis can be triggered during tumor development by a variety ofsignals. Extracellular signals include growth or survival factordepletion, hypoxia and ionizing radiation. Internal signals that cantrigger apoptosis include DNA damage, shortening telomeres, andoncogenic mutations that produce inappropriate proliferative signals.Lowe & Lin (2000) Carcinogenesis, 21, 485-495. Ionizing radiation andnearly all cytotoxic chemotherapy agents used to treat malignancies arethought to act by triggering endogenous apoptotic mechanisms to inducecell death. Rowan & Fisher (1997) Leukemia, 11, 457-465; Kerr et al.(1994) Cancer, 73, 2013-2026; Martin & Schwartz (1997) OncologyResearch, 9,1-5.

Evidence would suggest that early in the progression of cancer, tumorcells are sensitive to agents (such as ionizing radiation orchemotherapeutic drugs) that induce apoptosis. However, as the tumorprogresses, the cells develop resistance to apoptotic stimuli. Naik etal. (1996) Genes and Development, 10, 2105-2116. This may explain whyearly cancers respond better to treatment than more advanced lesions.The ability of late-stage cancers to develop resistance to chemotherapyand radiation therapy appears to be linked to alterations in theapoptotic pathway that limit the ability of tumor cells to respond toapoptotic stimuli. Reed et al. (1996) Journal of Cellular Biology, 60,23-32; Meyn et al. (1996) Cancer Metastasis Reviews, 15, 119-131; Hannun(1997) Blood, 89, 1845-1853; Reed (1995) Toxicology Letters, 82-83,155-158; Hickman (1996) European Journal of Cancer, 32A, 921-926.Resistance to chemotherapy has been correlated to overexpression of theanti-apoptotic gene bcl-2 and deletion or mutation of the pro-apoptoticbax gene in chronic lymphocytic leukemia and colon cancer, respectively.

The ability of tumor cells to successfully establish disseminatedmetastases also appears to involve alterations in the apoptotic pathway.Bold et al. (1997) Surgical Oncology, 6, 133-142. For example, mutationsin the tumor suppressor gene p53 are thought to occur in 70% of tumors.Evan et al. (1995) Curr. Opin. Cell Biol., 7, 825-834. Mutations thatinactivate p53 limit the ability of cells to induce apoptosis inresponse to DNA damage, leaving the cell vulnerable to furthermutations. Ko & Prives (1996) Genes and Development, 10, 1054-1072.

Therefore, apoptosis is intimately involved in the development andprogression of neoplastic transformation and metastases, and a betterunderstanding of the apoptotic pathways involved may lead to newpotential targets for the treatment of cancer by the modulation ofapoptotic pathways through gene therapy approaches. Bold et al. (1997)Surgical Oncology, 6, 133-142.

Deoxyhypusine synthase (DHS) and hypusine-containing eucaryotictranslation initiation Factor-5A (eIF-5A) are known to play importantroles in many cellular processes including cell growth anddifferentiation. Hypusine, a unique amino acid, is found in all examinedeucaryotes and archaebacteria, but not in eubacteria, and eIF-5A is theonly known hypusine-containing protein. Park (1988) J. Biol. Chem., 263,7447-7449; Schümann & Klink (1989) System. Appl. Microbiol., 11,103-107; Bartig et al. (1990) System. Appl. Microbiol., 13, 112-116;Gordon et al. (1987a) J. Biol. Chem., 262, 16585-16589. Active eIF-5A isformed in two post-translational steps: the first step is the formationof a deoxyhypusine residue by the transfer of the 4-aminobutyl moiety ofspermidine to the α-amino group of a specific lysine of the precursoreIF-5A catalyzed by deoxyhypusine synthase; the second step involves thehydroxylation of this 4-aminobutyl moiety by deoxyhypusine hydroxylaseto form hypusine.

The amino acid sequence of eIF-5A is well conserved between species, andthere is strict conservation of the amino acid sequence surrounding thehypusine residue in eIF-5A, which suggests that this modification may beimportant for survival. Park et al. (1993) Biofactors, 4, 95-104. Thisassumption is further supported by the observation that inactivation ofboth isoforms of eIF-5A found to date in yeast, or inactivation of theDHS gene, which catalyzes the first step in their activation, blockscell division. Schnier et al. (1991) Mol. Cell. Biol., 11, 3105-3114;Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J.Biol. Chem., 273, 1677-1683. However, depletion of eIF-5A protein inyeast resulted in only a small decrease in total protein synthesissuggesting that eIF-5A may be required for the translation of specificsubsets of mRNA's rather than for protein global synthesis. Kang et al.(1993), “Effect of initiation factor eIF-5A depletion on cellproliferation and protein synthesis,” in Tuite, M. (ed.), ProteinSynthesis and Targeting in Yeast, NATO Series H. The recent finding thatligands that bind eIF-5A share highly conserved motifs also supports theimportance of eIF-5A. Xu & Chen (2001) J. Biol. Chem., 276, 2555-2561.In addition, the hypusine residue of modified eIF-5A was found to beessential for sequence-specific binding to RNA, and binding did notprovide protection from ribonucleases.

The first cDNA for eIF-5A was cloned from human in 1989 by Smit-McBrideet al., and since then cDNAs or genes for eIF-5A have been cloned fromvarious eukaryotes including yeast, rat, chick embryo, alfalfa, andtomato. Smit-McBride et al. (1989a) J. Biol. Chem., 264, 1578-1583;Schnier et al. (1991) (yeast); Sano, A. (1995) in Imahori, M. et al.(eds), Polyamines, Basic and Clinical Aspects, VNU Science Press, TheNetherlands, 81-88 (rat); Rinaudo & Park (1992) FASEB J., 6, A453 (chickembryo); Pay et al. (1991) Plant Mol. Biol., 17, 927-929 (alfalfa); Wanget al. (2001) J. Biol. Chem., 276, 17541-17549 (tomato).

In addition, intracellular depletion of eIF-5A resulted in a significantaccumulation of specific mRNAs in the nucleus, indicating that eIF-5Amay be responsible for shuttling specific classes of mRNAs from thenucleus to the cytoplasm. Liu & Tartakoff (1997) Supplement to MolecularBiology of the Cell, 8, 426a. Abstract No. 2476, 37^(th) AmericanSociety for Cell Biology Annual Meeting. The accumulation of eIF-5A atnuclear pore-associated intranuclear filaments and its interaction witha general nuclear export receptor further suggest that eIF-5A is anucleocytoplasmic shuttle protein, rather than a component of polysomes.Rosorius et al. (1999) J. Cell Science, 112, 2369-2380.

Expression of eIF-5A mRNA has been explored in various human tissues andmammalian cell lines. For example, changes in eIF-5A expression havebeen observed in human fibroblast cells after addition of serumfollowing serum deprivation. Pang & Chen (1994) J. Cell Physiol., 160,531-538. Age-related decreases in deoxyhypusine synthase activity andabundance of precursor eIF-5A have also been observed in senescingfibroblast cells, although the possibility that this reflects averagingof differential changes in isoforms was not determined. Chen & Chen(1997b) J. Cell Physiol., 170, 248-254.

Studies have shown that eIF-5A may be the cellular target of viralproteins such as the human immunodeficiency virus type 1 Rev protein andhuman T cell leukemia virus type 1 Rex protein. Ruhl et al. (1993) J.Cell Biol.,123, 1309-1320; Katahira et al. (1995) J. Virol., 69,3125-3133. Preliminary studies indicate that eIF-5A may target RNA byinteracting with other RNA-binding proteins such as Rev, suggesting thatthese viral proteins may recruit eIF-5A for viral RNA processing. Liu etal. (1997) Biol. Signals, 6, 166-174.

Deoxyhypusine synthase and eIF-5A are known to play important roles inkey cellular processes including cell growth and senescence. Forexample, antisense reduction of deoxyhypusine synthase expression inplants results in delayed senescence of leaves and fruits, indicatingthat there is a senescence-inducing isoform of eIF-5A in plants. See WO01/02592; PCT/US01/44505; U.S. application Ser. No. 09/909,796.Inactivation of the genes for deoxyhypusine synthase or eIF-5A in yeastresults in inhibition of cell division. Schnier et al. (1991) Mol. Cell.Biol., 11, 3105-3114; Sasaki et al. (1996) FEBS Lett., 384, 151-154;Park et al. (1998) J. Biol. Chem., 273, 1677-1683.

Spermidine analogs have been successfully used to inhibit deoxyhypusinesynthase in vitro, as well as to inhibit the formation of hypusine invivo, which is accompanied by an inhibition of protein synthesis andcell growth. Jakus et al. (1993) J. Biol. Chem., 268, 13151-13159; Parket al. (1994) J. Biol. Chem., 269, 27827-27832. Polyamines themselves,in particular putrescine and spermidine, also appear to play importantroles in cellular proliferation and differentiation. Tabor & Tabor(1984) Annu. Rev. Biochem., 53, 749-790; Pegg (1988) Cancer Res., 48,759-774. For example, yeast mutants in which the polyamine biosynthesispathway has been blocked are unable to grow unless provided withexogenous polyamines. Cohn et al. (1980) J. Bacteriol., 134, 208-213.

Polyamines have also been shown to protect cells from the induction ofapoptosis. For example, apoptosis of thymocytes has been blocked byexposure to spermidine and spermine, the mechanism of which appears tobe the prevention of endonuclease activation. Desiderio et al. (1995)Cell Growth Differ., 6, 505-513; Brune et al. (1991) Exp. Cell Res.,195, 323-329. In addition, exogenous polyamines have been shown torepress B cell receptor-mediated apoptosis as well as apoptosis in theunicellular parasite, Trypanosoma cruzi. Nitta et al. (2001) Exptl. CellRes., 265, 174-183; Piacenza et al. (2001) Proc. Natl. Acad. Sci., USA,98, 7301-7306. Low concentrations of spermine and spermidine have alsobeen observed to reduce the number of nerve cells lost during normaldevelopment of newborn rats, as well as protect the brain from neuronaldamage during cerebral ischaemia. Gilad et al. (1985) Brain Res., 348,363-366; Gilad & Gilad (1991) Exp. Neurol., 111, 349-355. Polyaminesalso inhibit senescence, a form of programmed cell death, of planttissues. Spermidine and putrescine have been shown to delay post-harvestsenescence of carnation flowers and detached radish leaves. Wang & Baker(1980) HortScience, 15, 805-806 (carnation flowers); Altman (1982)Physiol. Plant., 54, 189-193 (detached radish leaves).

In other studies, however, induction of apoptosis has been observed inresponse to exogenous polyamines. For example, human breast cancer celllines responded to a polyamine analogue by inducing apoptosis, andexcess putrescine has been shown to induce apoptosis in DH23A cells.McCloskey et al. (1995) Cancer Res., 55, 3233-3236; Tome et al. (1997)Biochem. J., 328, 847-854.

The results from these experiments with polyamines collectively suggestthat existence of specific isoforms of eIF-5A play a role in inductionof apoptosis. Specifically, the data are consistent with the view thatthere is an apoptosis-specific isoform of eIF-5A, which is activated byDHS. The fact that this DHS reaction requires spermidine is consistentwith the finding that polyamines have been shown to elicit activation ofcaspase, a key executor of apoptosis-related proteolysis. Stefanelli etal. (2000) Biochem. J., 347, 875-880; Stefanelli et al. (1999) FEBSLett., 451, 95-98. In a similar vein, inhibitors of polyamine synthesiscan delay apoptosis. Das et al. (1997) Oncol. Res., 9, 565-572; Monti etal. (1998) Life Sci., 72, 799-806; Ray et al. (2000) Am. J. Physiol.,278, C480-C489; Packham & Cleveland (1994) Mol. Cell Biol., 14,5741-5747.

The finding that exogenous polyamines both inhibit and promote apoptosiscan be explained by the fact that, depending upon the levels applied,they can either inhibit the DHS reaction leading to activation of eIF-5Aand hence impede apoptosis, or induce apoptosis by reason of beingtoxic. The finding that low concentrations of exogenous polyamines blockapoptosis in plant and animal systems is consistent with the fact thatlow concentrations of polyamines and their analogues act as competitiveinhibitors of the DHS reaction. Indeed, even exogenous spermidine, whichis a substrate for the DHS reaction, will impede the reaction throughsubstrate inhibition. Jakus et al. (1993) J. Biol. Chem., 268,13153-13159.

However, all polyamines, and their analogues, are toxic at highconcentrations and are able to induce apoptosis. This occurs despitetheir ability to inhibit activation of the putative apoptosis-specificisoform of eIF-5A for two reasons. First, activated eIF-5A has a longhalf-life. Torrelio et al. (1987) Biochem. Biophys. Res. Commun., 145,1335-1341; Dou & Chen (1990) Biochim. Biophys. Acta., 1036, 128-137.Accordingly, depletion of activated apoptosis-specific eIF-5A arisingfrom inhibition of deoxyhypusine synthase activity may not occur in timeto block apoptosis caused by the toxic effects of spermidine. Second,polyamines are competitive inhibitors of the deoxyhypusine reaction andhence not likely to completely block the reaction even at concentrationsthat are toxic.

The present invention relates to cloning of an eIF-5A cDNA that is upregulated immediately before the induction of apoptosis. Thisapoptosis-specific eIF-5A is likely to be a suitable target forintervention in apoptosis-causing disease states since it appears to actat the level of post-transcriptional regulation of downstream effectorsand transcription factors involved in the apoptotic pathway.Specifically, the apoptosis-specific eIF-5A appears to selectivelyfacilitate the translocation of mRNAs encoding downstream effectors andtranscription factors of apoptosis from the nucleus to the cytoplasm,where they are subsequently translated. The ultimate decision toinitiate apoptosis appears to stem from a complex interaction betweeninternal and external pro- and anti-apoptotic signals. Lowe & Lin (2000)Carcinogenesis, 21, 485-495. Through its ability to facilitate thetranslation of downstream apoptosis effectors and transcription factors,the apoptosis-related eIF-5A appears to tip the balance between thesesignals in favor of apoptosis.

As described previously, it is well established that anticancer agentsinduce apoptosis and that alterations in the apoptotic pathways canattenuate drug-induced cell death. Schmitt & Lowe (1999) J. Pathol.,187, 127-137. For example, many anticancer drugs upregulate p53, andtumor cells that have lost p53 develop resistance to these drugs.However, nearly all chemotherapy agents can induce apoptosisindependently of p53 if the dose is sufficient, indicating that even indrug-resistant tumors, the pathways to apoptosis are not completelyblocked. Wallace-Brodeur & Lowe (1999) Cell Mol. Life Sci., 55, 64-75.This suggests that induction of apoptosis eIF-5A, even though it may notcorrect the mutated gene, may be able to circumvent the p53-dependentpathway and induce apoptosis by promoting alternative pathways.

Induction of apoptosis-related eIF-5A has the potential to selectivelytarget cancer cells while having little or no effect on normalneighboring cells. This arises because mitogenic oncogenes expressed intumor cells provide an apoptotic signal in the form of specific speciesof mRNA that are not present in normal cells. Lowe et al. (1993) Cell,74, 954-967; Lowe & Lin (2000) Carcinogenesis, 21, 485-495. For example,restoration of wild-type p53 in p53-mutant tumor cells can directlyinduce apoptosis as well as increase drug sensitivity in tumor celllines and xenographs. (Spitz et al., 1996; Badie et al., 1998).

The selectivity of apoptosis-eIF-5A arises from the fact that itselectively facilitates translation of mRNAs for downstream apoptosiseffectors and transcription factors by mediating their translocationfrom the nucleus into the cytoplasm. Thus, for apoptosis eIF-5A to havean effect, mRNAs for these effectors and transcription factors have tobe transcribed. Inasmuch as these mRNAs would be transcribed in cancercells, but not in neighboring normal cells, it is to be expected thatapoptosis eIF-5A would promote apoptosis in cancer cells but haveminimal, if any, effect on normal cells. Thus, restoration of apoptoticpotential in tumor cells with apoptosis-related eIF-5A may decrease thetoxicity and side effects experienced by cancer patients due toselective targeting of tumor cells. Induction of apoptotic eIF-5A alsohas the potential to potentiate the response of tumor cells toanti-cancer drugs and thereby improve the effectiveness of these agentsagainst drug-resistant tumors. This in turn could result in lower dosesof anti-cancer drugs for efficacy and reduced toxicity to the patient.

SUMMARY OF INVENTION

The present invention provides isolated and/or purified ratapoptosis-specific eIF-5A and DHS nucleic acids and polypeptides andantisense oligonucleotides and expression vectors of apoptosis-specificeIF-5A and DHS. The present invention also provides methods ofmodulating apoptosis using apoptosis-specific eIF-5A and DHS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence (SEQ ID NO:11) and derived aminoacid sequence of the 3′ end of rat apoptosis-specific eIF-5A (SEQ IDNO:12).

FIG. 2 depicts the nucleotide sequence (SR) ID NO:15) and derived aminoacid sequence of the 5′ end of rat apoptosis-specific eIF-5A cDNA (SEQID NO:16).

FIG. 3 depicts the nucleotide sequence of rat corpus luteumapoptosis-specific eIF-5A full length cDNA (SEQ ID NO:1) and derivedamino acid sequence (SEQ ID NO:2).

FIG. 4 depicts the nucleotide sequence (SEQ ID NO:6) and derived aminoacid sequence of the 3′ end of rat apoptosis-specific DHS cDNA (SEQ IDNO:7).

FIG. 5 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO:25) with thenucleotide sequence of human eIF-5A (Accession number BC000751 orNM_(—)001970, SEQ ID NO:3).

FIG. 6 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO:25) with thenucleotide sequence of human eIF-5A (Accession number NM-020390, SEQ IDNO:4).

FIG. 7 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO:25 with thenucleotide sequence of mouse eIF-5A (Accession number BC003889). Mousenucleotide sequence (Accession number BC003889) is SEQ ID NO:5.

FIG. 8 is an alignment of the derived full-length amino acid sequence ofrat corpus luteum apoptosis-specific eIF-5A (SEQ ID NO:2) with thederived amino acid sequence of human eIF-5A (Accession number BC000751or NM_(—)001970) (SEQ ID NO:26).

FIG. 9 is an alignment of the full-length amino acid sequence of ratcorpus luteum apoptosis-specific eIF-5A (SEQ ID NO:2) with the derivedamino acid sequence of human eIF-5A (Accession number NM_(—)020390) (SEQID NO:27).

FIG. 10 is an alignment of the derived full-length amino acid sequenceof rat corpus luteum apoptosis-specific eIF-5A (SEQ ID NO:2) with thederived amino acid sequence of mouse eIF-5A (Accession number BC003889)(SEQ ID NO:28).

FIG. 11 is an alignment of the partial-length nucleotide sequence of ratcorpus luteum apoptosis-specific DHS cDNA (SEQ ID NO:29) with thenucleotide sequence of human DHS (Accession number BC000333, SEQ IDNO8).

FIG. 12 is a restriction map of rat corpus luteum apoptosis-specificeIF-5A cDNA.

FIG. 13 is a restriction map of the partial-length ratapoptosis-specific DHS cDNA.

FIG. 14 is a Northern blot (FIG. 14A) and an ethidium bromide stainedgel (FIG. 14B) of total RNA probed with the ³²P-dCTP-labeled 3′-end ofrat corpus luteum apoptosis-specific eIF-5A cDNA.

FIG. 15 is a Northern blot (FIG. 15A) and an ethidium bromide stainedgel (FIG. 15B) of total RNA probed with the ³²P-dCTP-labeled 3′-end ofrat corpus luteum apoptosis-specific DHS cDNA.

FIG. 16 depicts a DNA laddering experiment in which the degree ofapoptosis in superovulated rat corpus lutea was examined after injectionwith PGF-2α.

FIG. 17 is an agarose gel of genomic DNA isolated from apoptosing ratcorpus luteum showing DNA laddering after treatment of rats with PGF-2α.

FIG. 18 depicts a DNA laddering experiment in which the degree ofapoptosis in dispersed cells of superovulated rat corpora lutea wasexamined in rats treated with spermidine prior to exposure toprostaglandin F-2α (PGF-2α).

FIG. 19 depicts a DNA laddering experiment in which the degree ofapoptosis in superovulated rat corpus lutea was examined in rats treatedwith spermidine and/or PGF-2α.

FIG. 20 is a Southern blot of rat genomic DNA probed with³²P-dCTP-labeled partial-length rat corpus luteum apoptosis-specificeIF-5A cDNA.

FIG. 21 depicts pHM6, a mammalian epitope tag expression vector (RocheMolecular Biochemicals).

FIG. 22 is a Northern blot (FIG. 22A) and ethidium bromide stained gel(FIG. 22B) of total RNA isolated from COS-7 cells after induction ofapoptosis by withdrawal of serum probed with the ³²P-dCTP-labeled3′-untranslated region of rat corpus luteum apoptosis-specific DHS cDNA.

FIG. 23 is a flow chart illustrating the procedure for transienttransfection of COS-7 cells.

FIG. 24 is a Western blot of transient expression of foreign proteins inCOS-7 cells following transfection with pHM6.

FIG. 25 illustrates enhanced apoptosis as reflected by increased caspaseactivity when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 26 illustrates enhanced apoptosis as reflected by increased DNAfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 27 illustrates detection of apoptosis as reflected by increasednuclear fragmentation when COS-7 cells were transiently transfected withpHM6 containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 28 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 29 illustrates detection of apoptosis as reflected byphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 30 illustrates enhanced apoptosis as reflected by increasedphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 31 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 32 illustrates enhanced apoptosis when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 33 illustrates down-regulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. FIG. 33A is theCoomassie-blue-stained protein blot; FIG. 33B is the correspondingWestern blot.

FIG. 34 is a Coomassie-blue-stained protein blot (FIG. 34A) and thecorresponding Western blot (FIG. 34B) of COS-7 cells transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the antisense orientation using Bcl-2 as a probe.

FIG. 35 is a Coomassie-blue-stained protein blot (FIG. 35A) and thecorresponding Western blot (FIG. 35B) of COS-7 cells transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation using c-Myc as a probe.

FIG. 36 is a Coomassie-blue-stained protein blot (FIG. 36A) and thecorresponding Western blot (FIG. 36B) of COS-7 cells transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation when p53 is used as a probe.

FIG. 37 is a Coomassie-blue-stained protein blot (FIG. 37A) and thecorresponding Western blot (FIG. 37B) of expression of pHM6-full-lengthrat apoptosis-specific eIF-5A in COS-7 cells using ananti-[HA]-peroxidase probe and a Coomassie-blue-stained protein blot(FIG. 37C) of expression of pHM6-full-length rat apoptosis-specificeIF-5A in COS-7 cells when a p53 probe is used.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery andcharacterization of a full-length cDNA encoding an eIF-5A isolated fromrat corpus luteum, which is involved in apoptosis (apoptosis-specific).Therefore, in one embodiment, the present invention provides an isolatednucleic acid comprising a nucleotide sequence encoding a ratapoptosis-specific eIF-5A polypeptide. Also provided by the presentinvention is a purified polypeptide comprising an amino acid sequence ofa rat apoptosis-specific eIF-5A polypeptide. Rat apoptosis-specificeIF-5A polypeptide means any polypeptide specific to rats that isdifferentially expressed in apoptosing cells and that results fromformation of a deoxyhypusine residue by the transfer of the 4-aminobutylmoiety of spermidine to the α-amino group of a specific conserved lysineof a precursor eIF-5A catalyzed by deoxyhypusine synthase andhydroxylation of this 4-aminobutyl moiety by deoxyhypusine hydroxylaseto form hypusine, thereby activating eIF-5A.

In addition, the nucleic acid and polypeptide rat apoptosis-specificeIF-5A sequences of the present invention can be used to isolateapoptosis-specific nucleic acids and polypeptides from other cells,tissues, organs, or animals using guidance provided herein andtechniques well known to those skilled in the art. The present inventionalso provides nucleic acid molecules that are suitable as primers orhybridization probes for the detection of nucleic acids encoding a ratapoptosis-specific eIF-5A polypeptide of the invention.

The nucleic acids of the present invention can be DNA, RNA, DNA/RNAduplexes, protein-nucleic acid (PNA), or derivatives thereof. As usedherein, a nucleic acid or polypeptide is said to be “isolated” or“purified” when it is substantially free of cellular material or free ofchemical precursors or other chemicals. It should be appreciated thatthe term isolated or purified does not refer to a library-typepreparation containing a myriad of other sequence fragments. The nucleicacid or polypeptide of the present invention can be purified tohomogeneity or other degrees of purity. The level of purification willbe based on the intended use. The critical feature is that thepreparation allows for the desired function of the nucleic acid orpolypeptide, even if in the presence of considerable amounts of othercomponents.

The isolated polypeptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods. Forexample recombinant production of proteins involves cloning a nucleicacid molecule encoding either the apoptosis inducing eIF-5A or DHS intoan expression vector. The expression vector is introduced into a hostcell and the protein is expressed in the host cell. The protein can thenbe isolated from the cells by any appropriate purification scheme usingstandard protein purification techniques.

Preferably, the isolated nucleic acid encoding a rat apoptosis-specificeIF-5A polypeptide of the present invention has a nucleotide sequence ofSEQ ID NO:1 and the purified polypeptide of the present invention has anamino acid sequence of SEQ ID NO:2. The present inventive ratapoptosis-specific eIF-5A nucleic acids and polypeptides also encompasssequences that have substantial sequence identity or homology to SEQ IDNO:1 and SEQ ID NO:2, respectively, as well as functional derivativesand variants thereof.

As used herein, the term “substantial sequence identity” or “substantialhomology” is used to indicate that a sequence exhibits substantialstructural or functional equivalence with another sequence. Anystructural or functional differences between sequences havingsubstantial sequence identity or substantial homology will be deminimus; that is, they will not affect the ability of the sequence tofunction as indicated in the desired application. Differences may be dueto inherent variations in codon usage among different species, forexample. Structural differences are considered de minimus if there is asignificant amount of sequence overlap or similarity between two or moredifferent sequences or if the different sequences exhibit similarphysical characteristics even if the sequences differ in length orstructure. Such characteristics include, for example, the ability tohybridize under defined conditions, or in the case of proteins,immunological crossreactivity, similar enzymatic activity, etc. Theskilled practitioner can readily determine each of these characteristicsby art known methods.

Additionally, two nucleotide sequences are “substantially complementary”if the sequences have at least about 70 percent or greater, morepreferably 80 percent or greater, even more preferably about 90 percentor greater, and most preferably about 95 percent or greater sequencesimilarity between them. Two amino acid sequences are substantiallyhomologous if they have at least 50%, preferably at least 70%, morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 95% similarity between the active, or functionallyrelevant, portions of the polypeptides.

To determine the percent identity of two sequences, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). In a preferred embodiment, at least 30%, 40%,50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequenceis aligned for comparison purposes. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram. BLAST protein searches can be performed with the XBLAST programto obtain amino acid sequences homologous to the proteins of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

The term “functional derivative” of a nucleic acid is used herein tomean a homolog or analog of the gene or nucleotide sequence. Afunctional derivative may retain at least a portion of the function ofthe given gene, which permits its utility in accordance with theinvention. “Functional derivatives” of the apoptosis-specific eIF-5Apolypeptide as described herein are fragments, variants, analogs, orchemical derivatives of apoptosis-specific eIF-5A that retain at least aportion of the apoptosis-specific eIF-5A activity or immunological crossreactivity with an antibody specific for apoptosis-specific eIF-5A. Afragment of the apoptosis-specific eIF-5A polypeptide refers to anysubset of the molecule.

Functional variants can also contain substitutions of similar aminoacids that result in no change or an insignificant change in function.Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al. (1989) Science 244:1081-1085). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et al.(1992) Science 255:306-312).

A “variant” refers to a molecule substantially similar to either theentire gene or a fragment thereof, such as a nucleotide substitutionvariant having one or more substituted nucleotides, but which maintainsthe ability to hybridize with the particular gene or to encode mRNAtranscript which hybridizes with the native DNA. A “homolog” refers to afragment or variant sequence from a different animal genus or species.An “analog” refers to a non-natural molecule substantially similar to orfunctioning in relation to the entire molecule, a variant or a fragmentthereof.

Variant peptides include naturally occurring variants as well as thosemanufactured by methods well known in the art. Such variants can readilybe identified/made using molecular techniques and the sequenceinformation disclosed herein. Further, such variants can readily bedistinguished from other proteins based on sequence and/or structuralhomology to the eIF-5A or DHS proteins of the present invention. Thedegree of homology/identity present will be based primarily on whetherthe protein is a functional variant or non-functional variant, theamount of divergence present in the paralog family and the evolutionarydistance between the orthologs.

Non-naturally occurring variants of the eIF-5A or DHS proteins of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the proteins. For example,one class of substitutions are conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in a proteinby another amino acid of like characteristics. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu, and Ile; interchange of thehydroxyl residues Ser and Thr; exchange of the acidic residues Asp andGlu; substitution between the amide residues Asn and Gln; exchange ofthe basic residues Lys and Arg; and replacements among the aromaticresidues Phe and Tyr. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247:1306-1310 (1990).

Alternatively, but also preferably, the nucleic acid encoding a ratapoptosis-specific eIF-5A polypeptide of the present inventionhybridizes under highly stringent conditions with a nucleotide sequencethat is complementary to that of SEQ ID NO:1. The term “hybridization”as used herein is generally used to mean hybridization of nucleic acidsat appropriate conditions of stringency as would be readily evident tothose skilled in the art depending upon the nature of the probe sequenceand target sequences. Conditions of hybridization and washing are wellknown in the art, and the adjustment of conditions depending upon thedesired stringency by varying incubation time, temperature and/or ionicstrength of the solution are readily accomplished. See, e.g. Sambrook,J. et al., Molecular Cloning: A Laboratory Manual, 2^(nd) edition, ColdSpring Harbour Press, Cold Spring Harbor, N.Y., 1989.

The choice of conditions is dictated by the length of the sequencesbeing hybridized, in particular, the length of the probe sequence, therelative G-C content of the nucleic acids and the amount of mismatchesto be permitted. Low stringency conditions are preferred when partialhybridization between strands that have lesser degrees ofcomplementarity is desired. When perfect or near perfect complementarityis desired, high stringency conditions are preferred. For typical highstringency conditions, the hybridization solution contains 6×S.S.C.,0.01 M EDTA, 1× Denhardt's solution and 0.5% SDS. Hybridization iscarried out at about 68° C. for about 3 to 4 hours for fragments ofcloned DNA and for about 12 to 16 hours for total eucaryotic DNA. Forlower stringencies, the temperature of hybridization is reduced to about42° C. below the melting temperature (T_(m)) of the duplex. The T_(m) isknown to be a function of the G-C content and duplex length as well asthe ionic strength of the solution.

As used herein, the phrase “hybridizes to a corresponding portion” of aDNA or RNA molecule means that the molecule that hybridizes, e.g.,oligonucleotide, polynucleotide, or any nucleotide sequence (in sense orantisense orientation) recognizes and hybridizes to a sequence inanother nucleic acid molecule that is of approximately the same size andhas enough sequence similarity thereto to effect hybridization underappropriate conditions. For example, a 100 nucleotide long sensemolecule will recognize and hybridize to an approximately 100 nucleotideportion of a nucleotide sequence, so long as there is about 70% or moresequence similarity between the two sequences. It is to be understoodthat the size of the “corresponding portion” will allow for somemismatches in hybridization such that the “corresponding portion” may besmaller or larger than the molecule which hybridizes to it, for example20-30% larger or smaller, preferably no more than about 12-15% larger orsmaller.

In addition, functional variants of polypeptides can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Amino acids that are essential forfunction can be identified by methods known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunningham etal., Science 244:1081-1085 (1989)). The latter procedure introducessingle alanine mutations at every residue in the molecule. The resultingmutant molecules are then tested for biological activity or in assays.

For example, an analog of apoptosis-specific eIF-5A refers to anon-natural protein or peptidomimetic substantially similar to eitherthe entire protein or a fragment thereof. Chemical derivatives ofapoptosis-specific eIF-5A contain additional chemical moieties notnormally a part of the peptide or peptide fragment. Modifications can beintroduced into peptide or fragment thereof by reacting targeted aminoacid residues of the peptide with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues.

The initial discovery and characterization of a full-length cDNAencoding an apoptosis-specific eIF-5A isolated from rat corpus luteumled to the discovery and characterization of a partial-length cDNA cloneencoding a DHS, which is also isolated from rat corpus luteum andinvolved in apoptosis. Accordingly, in an additional embodiment, thepresent invention provides an isolated nucleic acid comprising anucleotide sequence encoding a rat apoptosis-specific DHS polypeptide.Also provided is a purified polypeptide comprising an amino acidsequence of a rat apoptosis-specific DHS polypeptide. Ratapoptosis-specific DHS polypeptide means any suitable polypeptidespecific to rats that is differentially expressed in apoptosing cellsand that catalyzes formation of a deoxyhypusine residue by the transferof the 4-aminobutyl moiety of spermidine to the α-amino group of aspecific conserved lysine of inactive eIF-5A to form deoxyhypusine,thereby activating eIF-5A.

Preferably, the isolated nucleic acid encoding a rat apoptosis-specificDHS polypeptide of the present invention has a nucleotide sequence ofSEQ ID NO:6 and the purified polypeptide of the present invention has anamino acid sequence of SEQ ID NO:7. The present inventive ratapoptosis-specific DHS nucleic acids and polypeptides also encompasssequences that have substantial sequence identity or homology to SEQ IDNO:6 and SEQ ID NO:7, respectively, as well as functional derivativesand variants thereof, which have been described previously.Alternatively, and also preferably, the isolated nucleic acid of thepresent invention has a nucleotide sequence that hybridizes under highlystringent conditions with the complement of SEQ ID NO:6, which also hasbeen described previously.

As is the case with the nucleic acids and polypeptides of the ratapoptosis-specific eIF-5A sequences described herein, the nucleic acidsand polypeptides of the rat apoptosis-specific DHS sequences of thepresent invention can be used to isolate apoptosis-specific DHS nucleicacids and polypeptides from other animals, including humans. Isolationof such DHS sequences from animals and human can be achieved using artknown methods and guidance provided herein, based on sequencesimilarities of at least 80% across species. The present invention alsoprovides nucleic acid molecules that are suitable as primers orhybridization probes for the detection of nucleic acids encoding a ratapoptosis-specific DHS polypeptide of the invention.

Apoptosis-specific eIF-5A and DHS are suitable targets for regulation ofapoptosis, including apoptosis underlying disease processes, since itlikely acts in the post-transcriptional regulation of downstreameffectors and transcription factors involved in the apoptotic pathway.Thus, the present invention also provides methods of modulatingapoptosis in a cell by administering to the cell an agent that modulatesapoptosis-specific eIF-5A and/or DHS function. It should be appreciatedby one of skill in the art that the agent can be one that modulates onlyapoptosis-specific eIF-5A function, only apoptosis-specific DHS functionalone, or both apoptosis-specific eIF-5A and DHS function.

Apoptosis can be modulated by any suitable alteration in the normallevel of apoptosis-specific eIF-5A and/or DHS function in the cell. Asintended herein, modification or alteration can be complete or partialand can include a change in transcriptional or translational control orother change altering apoptosis-specific eIF-5A and/or DHS function inthe cell. Apoptosis-specific eIF-5A or DHS function means any activityrelating to formation of a deoxyhypusine residue by the transfer of the4-aminobutyl moiety of spermidine to the α-amino group of a specificconserved lysine of a precursor eIF-5A, which is catalyzed by DHS, andhydroxylation of this 4-aminobutyl moiety by deoxyhypusine hydroxylaseto form hypusine, thereby activating eIF-5A.

In one embodiment of the present invention, the agent can inhibitapoptosis-specific eIF-5A and/or DHS function, thereby inhibitingapoptosis. Inhibiting apoptosis means any decrease, in intensity and/ornumber, and/or delay in onset of any or all of the well-definedmorphological features characteristic of apoptosis, such as, forexample, cell shrinkage, chromatin condensation, nuclear fragmentation,and membrane blebbing.

One agent that can inhibit apoptosis-specific eIF-5A and/or DHS functionis an antisense oligonucleotide. Preferably, the antisenseoligonucleotide has a nucleotide sequence encoding a portion of anapoptosis-specific eIF-5A polypeptide and/or an apoptosis-specific DHSpolypeptide. Many suitable nucleic acid sequences encoding anapoptosis-specific eIF-5A polypeptide and/or DHS polypeptide are knownin the art. For example, SEQ ID NOS:1, 3, 4, 5, 11, 15, 19, 20, and 21(apoptosis-specific eIF-5A nucleic acid sequences), SEQ ID NOS:6 and 8(apoptosis-specific DHS nucleic acid sequences), SEQ ID NOS: 12 and 16eIF-5A (apoptosis-specific polypeptide sequences), and SEQ ID NO:7(apoptosis-specific DHS polypeptide sequences), or portions thereof,provide suitable sequences. Others suitable sequences can be found usingthe known sequences as probes according to the methods described herein.

Accordingly, the present invention also provides antisenseoligonucleotides encoding a portion of an apoptosis-specific eIF-5Apolypeptide and/or an apoptosis-specific DHS polypeptide, or acomplement thereof. The antisense oligonucleotides of the presentinvention can be in the form of RNA or DNA, e.g., cDNA, genomic DNA, orsynthetic RNA or DNA. The DNA can be double-stranded or single stranded,and if single stranded can be the coding strand or non-coding strand.The specific hybridization of an oligomeric compound with its targetnucleic acid, resulting in interference with the normal function of thenucleic acid, is generally referred to as “antisense.” The functions ofDNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include all functions such as,for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which can beengaged in or facilitated by the RNA. The overall effect of suchantisense oligonucleotide is inhibiting of expression ofapoptosis-specific eIF-5A and/or DHS and/or the amount of activatedapoptosis-specific eIF-5A produced.

Alternatively, the activation of apoptosis-specific eIF-5A byapoptosis-specific DHS can be inhibited by administering chemical agentsthat inhibit the DHS enzymatic reaction. For example, the onset of DNAladdering reflecting apoptosis is delayed in rat corpus luteum when theanimals are treated with spermidine, an inhibitor of the DHS reactionafter induction of apoptosis by injection of PGF-2α (FIGS. 18-19). Jakuset al., (1993) J. Biol. Chem. 268: 13151-13159.

Apoptosis also can be inhibited or substantially decreased by addingagents that degrade apoptosis-specific eIF-5A DNA, RNA, or protein, orthat degrade apoptosis-specific DHS DNA, RNA, or protein, therebypreventing the activation of apoptosis-specific eIF-5A byapoptosis-specific DHS. In another embodiment of the invention,inhibition of expression of endogenous mammalian apoptosis-specific DHS,apoptosis-specific eIF-5A, or both, are affected through the use ofribozymes. Examples of suitable drugs include those that inhibit theactivation of apoptosis-specific eIF-5A by apoptosis-specific DHS, thosethat inhibit the activation of apoptosis-specific eIF-5A bydeoxyhypusine hydroxylase, those that inhibit transcription and/ortranslation of apoptosis-specific DHS, those that inhibit transcriptionand/or translation of apoptosis-specific deoxyhypusine hydroxylase, andthose that inhibit transcription or translation of apoptosis-specificeIF-5A. Examples of drugs that inhibit the activation of eIF-5A byapoptosis-specific DHS are spermidine, 1,3-Diamino-propane,1,4-Diamino-butane (putrescine), 1,7-Diamino-heptane, or1,8-Diamino-octane.

It is also possible to inhibit apoptosis-specific eIF-5A by inactivatingthe gene coding for apoptosis-specific eIF-5A in a cell. Suchinactivation can occur by deleting the gene in the cell or byintroducing a deletion or mutation into the gene and therebyinactivating the gene. The gene can also be inactivated by insertinginto the gene another DNA fragment such that expression of theendogenous apoptosis-specific eIF-5A protein does not occur. Likewise,it is possible to inhibit activation of apoptosis-specific eIF-5A byinactivating the gene coding for apoptosis-specific DHS in a cell.Methods for introducing mutations, such as deletions and insertions,into genes in eukaryotic cells are known in the art, e.g., U.S. Pat. No.5,464,764. Oligonucleotides and expression vectors useful for mutationof genes in cells can be made according to methods known in the art andguidance provided herein; for example, methods useful for making andexpressing antisense oligonucleotides can be used to makeoligonucleotides and expression vectors useful for mutating genes incells.

It is also possible to inhibit expression of apoptosis-specific eIF-5Aby suppressing expression of the gene coding for apoptosis-specificeIF-5A in a cell. Such inactivation can be accomplished viacosuppression, e.g., by introducing nucleotide sequence(s) coding forapoptosis-specific eIF-5A into a cell such that cosuppression occurs.Likewise, it is possible to inhibit activation of apoptosis-specificeIF-5A by suppressing the expression of the gene coding forapoptosis-specific DHS in a cell via cosuppression. Oligonucleotides andexpression vectors useful for cosuppression can be made according tomethods known in the art and guidance provided herein; for example,methods useful for making and expressing antisense oligonucleotides canbe used to make oligonucleotides and expression vectors useful forcosuppression. Methods for cosuppression are known in the art, e.g.,U.S. Pat. No. 5,686,649.

One result of the inhibition (through, e.g., antisense, mutation, orcosuppression) is a reduction in the amount of endogenous translatableapoptosis-specific eIF-5A or DHS-encoding mRNA. Consequently, the amountof apoptosis-specific DHS protein produced is reduced, thereby reducingthe amount of activated eIF-5A, which in turn reduces translation ofapoptosis-specific proteins. Apoptosis is thus inhibited or delayed,since de novo protein synthesis is required for the onset of apoptosis.

In another embodiment of the present invention, the agent can induceapoptosis-specific eIF-5A or DHS function, thereby inducing apoptosis.Inducing apoptosis means any increase, in intensity or number, oracceleration in onset of any or all of the well-defined morphologicalfeatures characteristic of apoptosis, such as, for example, cellshrinkage, chromatin condensation, nuclear fragmentation, and membraneblebbing.

Any suitable agent that induces apoptosis-specific eIF-5A and/or DHSfunction can be used. It is appreciated by one of skill in the art thatboth the inactive and active forms of apoptosis-specific eIF-5A can beadministered. If the inactive form, or hypusine-unmodified form, isadministered, native apoptosis-specific DHS will activate the eIF-5A.Many suitable nucleic acid sequences encoding an apoptosis-specificeIF-5A polypeptide and/or DHS polypeptide are known in the art. Forexample, SEQ ID NOS:1, 3, 4, 5, 11, 15, 19, 20, and 21(apoptosis-specific eIF-5A nucleic acid sequences), SEQ ID NOS:6 and 8(apoptosis-specific DHS nucleic acid sequences), SEQ ID NOS: 12 and 16eIF-5A (apoptosis-specific polypeptide sequences), and SEQ ID NO:7(apoptosis-specific DHS polypeptide sequences), or portions thereof,provide suitable sequences. Others suitable sequences can be found usingthe known sequences as probes according to the methods described herein.

For example, naked nucleic acids (naked DNA vectors such asoligonucleotides or plasmids), or polypeptides, including recombinantlyproduced polypeptides, can be administered to a cell. Recombinantlyproduced polypeptides means that the DNA sequences encoding the eIF-5Aor the DHS proteins are placed into a suitable expression vector, whichis described in detail below. The host is transfected with theexpression vector and thereafter produces the desired polypeptides. Thepolypeptides are then isolated from the host cell. Recombinantapoptosis-inducing eIF-5A protein can be made, for example, in ChineseHamster Ovary (CHO) cells and activated using recombinant DHS by thoseskilled in the art. Wang et al. (2001) J. Biol. Chem., 276, 17541-17549;Eriksson et al., (2001) Semin. Hematol., 38, 24-31. The polypeptides canalso be synthetic, which are synthesized using known protein synthesismethods

Polypeptide uptake can be facilitated using ligands, for example, aligand derived from anthrax that mediates uptake into a broad range ofcells. Liu et al. (2001) J. Biol. Chem., 276, 46326-46332. Recombinantprotein can also be administered to target cells, tissues, and organs ofmammals using liposomes. Liposomes occluding the protein areadministered intravenously. Targeting can be achieved by incorporatingligands to specific cell receptors into the liposomes. See, e.g.,Kaneda, Adv Drug Delivery Rev 43: 197-205 (2000).

One preferred agent that can induce induce apoptosis-specific eIF-5A orDHS function is an expression vector. Accordingly, the present inventionprovides expression vectors having a promoter operably linked to anucleic acid encoding an apoptosis-specific eIF-5A polypeptide and/orDHS polypeptide. The expression vectors of the present invention can bein the form of RNA or DNA, e.g., cDNA, genomic DNA, or synthetic RNA orDNA. The DNA can be double-stranded or single stranded, and if singlestranded can be the coding strand or non-coding strand. Any appropriateexpression vector (see, e.g., Pouwels et al., Cloning Vectors: ALaboratory Manual(Elsevior, N.Y.: 1985)) can be used. Preferably, theexpression vector has a promoter sequence operably linked to anucleotide sequence encoding an apoptosis-specific (related) eIF-5Apolypeptide and/or apoptosis-specific (related) DHS polypeptide.

Within the expression vector, the desired nucleic acid and the promoterare operably linked such that the promoter is able to drive theexpression of the nucleic acid. Any suitable promoter can be usedprovided that the nucleic acid is expressed. Examples of such suitablepromoters include various viral promoters, eucaryotic promoters, andconstitutively active promoters. As long as this operable linkage ismaintained, the expression vector can include more than one nucleic acid(e.g., nucleic acids encoding both apoptosis-specific eIF-5A and/orDHS). The expression vector can optionally include other elements, suchas polyadenylation sequences, ribosome entry sites, transcriptionalregulatory elements (e.g., enhancers, silencers, etc.), other sequencesfor enhancing the stability of the vector or transcript or thetranslation or processing of the desired transcript within the cells(e.g., secretion signals, leaders, etc.), or any other suitable element.

Expression vector can be derived from viruses such as adenovirus,adeno-associated virus, herpesvirus, retrovirus or lentivirus. Theexpression vector of the present invention can be transfected into hostcells, which include, but are not limited to, bacterial species,mammalian or insect host cell systems including baculovirus systems(see, e.g., Luckow et al., Bio/Technology, 6, 47 (1988)), andestablished cell lines such 293, COS-7, C127, 3T3, CHO, HeLa, BHK, etc.

Adenoviral vectors are preferred because, unlike plasmids and otherviral vectors (e.g., herpes simplex virus), adenoviral vectors achievegene transfer in both dividing and nondividing cells, with high levelsof protein expression in cardiovascular relevant sites such asmyocardium, vascular endothelium, and skeletal muscle. Furthermore, thegene transferred by an adenoviral vector functions in an epichromosomalposition and thus carries little risk of inappropriately inserting thetransferred gene into a critical site of the host genome. The adenoviralvector also desirably is deficient in at least one gene functionrequired for viral replication. Preferably, the adenoviral vector isdeficient in at least one essential gene function of the E1, E2, and/orE4 regions of the adenoviral genome. More preferably, the vectoradditionally is deficient in at least part of the E3 region of theadenoviral genome (e.g., an XbaI deletion of the E3 region).

Recombinant adenovirus can be delivered to cultured cells by simplyadding the virus to the culture media. Infection of host animals/humanscan be achieved by directly injecting the viral particles into thebloodstream or into the desired tissue. The half-life of the virus inserum can be extended by complexing the virus with liposomes (e.g.Lipofectin, Life Technologies) or polyethylene glycol. The adenovirusvector normally enters the cell through an interaction between the knobdomain of the viral fiber protein and the coxsackievirus and adenovirusreceptor, CAR. The viral vector can be directed to specific cells, or tocells which do not express the CAR, by genetically engineering the virusto express a ligand specific to a certain cell receptor.

In an alternate embodiment, apoptosis can be initiated or enhanced bychemically upregulating the transcription of endogenousapoptosis-specific eIF-5A, or apoptosis-specific DHS, or both, withchemicals, or by chemically enhancing the activation ofapoptosis-specific eIF-5A. In one such embodiment, PGF-2α isadministered to the cancer cells or tumor of the animal/human toupregulate the transcription of DHS and eIF-5A.

Apoptosis-specific eIF-5A is a suitable target for regulation ofapoptosis, including apoptosis underlying disease processes, since itlikely acts in the post-transcriptional regulation of downstreameffectors and transcription factors involved in the apoptotic pathway.The present inventive methods of modulating apoptosis-specific eIF-5Aand apoptosis-specific DHS, either alone or in combination, can beaccomplished in cells of animals resulting in induction or enhancementof apoptosis and giving rise to novel methods and compositions for thetreatment and prevention of diseases caused by, causing, or otherwisehaving an etiology associated with an inability of cells to undergoapoptosis.

Many important human diseases are caused by abnormalities in the controlof apoptosis. These abnormalities can result in either a pathologicalincrease in cell number (e.g. cancer) or a damaging loss of cells (e.g.degenerative diseases). As non-limiting examples, the methods andcompositions of the present invention can be used to prevent or treatthe following apoptosis-associated diseases and disorders:neurological/neurodegerative disorders (e.g., Alzheimer's, Parkinson's,Huntington's, Amyotrophic Lateral Sclerosis (Lou Gehrig's Disease),autoimmune disorders (e.g., rheumatoid arthritis, systemic lupuserythematosus (SLE), multiple sclerosis), Duchenne Muscular Dystrophy(DMD), motor neuron disorders, ischemia, chronic heart failure, stroke,infantile spinal muscular atrophy, cardiac arrest, renal failure, atopicdermatitis, sepsis and septic shock, AIDS, hepatitis, glaucoma, diabetes(type 1 and type 2), asthma, retinitis pigmentosa, osteoporosis,xenograft rejection, and burn injury.

The present inventive methods can be used for therapeutic treatments toan animal having a cancerous cell or suffering from a tumor in an amountsufficient to kill the cancerous cell or inhibit the progression of thetumor, respectively. An amount adequate to accomplish this is defined asa therapeutically effective dose. Amounts effective for this use willdepend upon the severity of the disease and the general state of theanimal's own immune system.

Inhibition of tumor growth means prevention or reduction of theprogression of the tumor, e.g, the growth, invasiveness, metastasesand/or recurrence of the tumor. The present inventive methods can beused to treat any suitable tumor, including, for example, tumors of thebreast, heart, lung, small intestine, colon, spleen, kidney, bladder,head and neck, ovary, prostate, brain, pancreas, skin, bone, bonemarrow, blood, thymus, uterus, testicles, cervix or liver. Animals,preferably mammals, and more preferably humans can be treated using thecompositions and methods of the present invention. The present inventivemethods can thus be carried out in vitro, ex vivo, or in vivo.

Dosing schedules will also vary with the disease state and status of theanimal, and will typically range from a single bolus dosage orcontinuous infusion to multiple administrations per day (e.g., every 4-6hours), or as indicated by the treatment and the animals's condition. Itshould be noted, however, that the present invention is not limited toany particular dose.

In the present invention, any suitable method or route can be used foradministration, for example, oral, intravenous, intraperitoneal,subcutaneous, or intramuscular administration. The dose of antagonistadministered depends on numerous factors, including, for example, thetype of molecule administered, the type and severity tumor being treatedand the route of administration. It should be emphasized, however, thatthe present invention is not limited to any particular method or routeof administration.

In one alternative embodiment, the present inventive methods can be usedin combination with one or more traditional therapies. For example, asuitable antineoplastic agent can be used, such as a chemotherapeuticagent or radiation. In an additional alternative embodiment, the presentinventive methods can be used in combination with one or more suitableadjuvants, such as, for example, cytokines (IL-10 and IL-13, forexample) or other immune stimulators.

In another alternative embodiment, diagnosis of an apoptosis-relateddisorder can be made using apoptosis-specific eIF-5A and proliferatingeIF-5A, which differs from the apoptosis-specific eIF-5A in that theyare transcribed from different locations by different promoters;although the two are structurally homologous, with differences in thecarboxy terminus. The method of diagnosis of the present inventioninvolves comparing the amount of proliferating eIF-5A present in a givencell with the amount of apoptosis-specific eIF-5A present in the samecell. During normal functioning, a cell will have a greater amount ofproliferating eIF-5A than apoptosis-specific eIF-5A. However, in somecancer cells, the normal regulatory mechanisms go awry and the amount ofapoptosis-specific eIF-5A relative to the amount of proliferating eIF-5Ais altered. This potentially allows for diagnosis of a cell as cancerousprior to any phenotypic changes in the cell.

In yet another embodiment, the ratio of proliferating eIF-5A toapoptosis-specific eIF-5A can be used in drug screening. Such a methodalso involves comparing the amount of proliferating eIF-5A present in agiven cell with the amount of apoptosis-specific eIF-5A present in thesame cell. The normal ratio of proliferating eIF-5A toapoptosis-specific eIF-5A would be compared to the ratio ofproliferating eIF-5A to apoptosis-specific eIF-5A after contacting thecell with the drug candidate. Alterations in the ratio of proliferatingeIF-5A to apoptosis-specific eIF-5A after contact allows foridentification of those candidates that have apoptosis-modulatingactivity. Candidates having apoptosis-modulating activity can be usefulin treating diseases associated with apoptosis, either throughinhibition or induction of apoptosis. In addition, alterations in theratio of proliferating eIF-5A to apoptosis-specific eIF-5A can be usedto modulate apoptosis, which may also be useful to treat any of theconditions described herein as relating to abnormal apoptosis.

Using this method a large number of potential candidates, i.e., alibrary can be effectively screened to identify members of the librarythat modulate apoptosis. Any candidate or library of candidates can bescreened using this method. For example, biological response modifiersthat have shown promise as apoptosis modulators, including monoclonalantibodies that alter signal transduction pathways, cytokines such asTRAIL (Apo2 ligand), ligands for retinoid/steroid family nuclearreceptors, and small-molecule compounds that bind and inhibit proteinkinases, can be screened for definite apoptosis-modulating activityusing the present methods.

One suitable candidate is a protein kinase C-alpha antisenseoligonucleotide, ISIS 3521 (ISIS Pharmaceuticals, Inc., Carlsbad,Calif.), which has anti-tumor activity. Other specific candidatesinclude caspases (Idun Pharmaceuticals, San Diego, Calif.), which areknown to play a crucial role in the triggering and execution ofapoptosis in a variety of cell types leading to cancer andneurodegenerative diseases; GENASENSE™ (Genta, Inc., Berkeley Heights,N.J.), which is an antisense drug that blocks the production of Bcl-2;INGN 241 (Introgen Therapeutics, Inc., Houston, Tex.), which is a genetherapy targeting P53; rituximab (IDEC Corporation, Osaka, Japan), whichis an anti-CD20 monoclonal antibody; and general apoptosis driventherapies for cardiovascular disease and cancer (

gera Therapeutics Inc., Quebec, Canada).

It is understood that the nucleic acids and polypeptides of the presentinvention, where used in an animal for the purpose of prophylaxis ortreatment, will be administered in the form of a compositionadditionally comprising a pharmaceutically acceptable carrier. Suitablepharmaceutically acceptable carriers include, for example, one or moreof water, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. Pharmaceuticallyacceptable carriers can further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the bindingproteins. The compositions of the injection can, as is well known in theart, be formulated so as to provide quick, sustained or delayed releaseof the active ingredient after administration to the mammal.

The compositions of this invention can be in a variety of forms. Theseinclude, for example, solid, semi-solid and liquid dosage forms, such astablets, pills, powders, liquid solutions, dispersions or suspensions,liposomes, suppositories, injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application.

Such compositions can be prepared in a manner well known in thepharmaceutical art. In making the composition the active ingredient willusually be mixed with a carrier, or diluted by a carrier, and/orenclosed within a carrier which can, for example, be in the form of acapsule, sachet, paper or other container. When the carrier serves as adiluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, excipient or medium for the active ingredient. Thus, thecomposition can be in the form of tablets, lozenges, sachets, cachets,elixirs, suspensions, aerosols (as a solid or in a liquid medium),ointments containing for example up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, injectionsolutions, suspensions, sterile packaged powders and as a topical patch.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration. The Examples are set forth to aidin understanding the invention but are not intended to, and should notbe construed to, limit its scope in any way. The examples do not includedetailed descriptions of conventional methods. Such methods are wellknown to those of ordinary skill in the art and are described innumerous publications. Detailed descriptions of conventional methods,such as those employed in the construction of vectors and plasmids, theinsertion of nucleic acids encoding polypeptides into such vectors andplasmids, the introduction of plasmids into host cells, and theexpression and determination thereof of genes and gene products can beobtained from numerous publication, including Sambrook, J. et al.,(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory Press. All references mentioned herein areincorporated in their entirety.

EXAMPLES Example 1

The present example demonstrates isolation and characterization of afull-length cDNA encoding a rat eIF-5A nucleic acid exhibitingapoptosis-specific expression.

Superovulation and Induction of Apoptosis in Rat Corpus Luteum

Immature (21-30 day old) female rats were superovulated by subcutaneousinjection with 50 IU of PMSG (Pregant Mare Serum Gonadotropin) and 60 to65 hours later with 50 IU of HCG (Human Chorionic Gonadotropin). Sevendays after the treatment with HCG, corpus luteum apoptosis was inducedby subcutaneous injection with 500 mg of PGF-2α. Rats were sacrificed atvarious times (e.g., 1, 8, and 24 hours) after PGF-2α treatment, and thecorpora lutea were removed and placed in liquid nitrogen. Control corpusluteum tissue was obtained by sacrificing rats immediately before PGF-2αtreatment.

Dispersion of Rat Ovarian Corpus Luteum Cells

Six to nine days after superovulation, rats were treated by multisitesubcutaneous injection with 500 mg PGF-2α. Fifteen to thirty minuteslater, the ovaries were removed from the superovulated rats, placed inEBSS (Gibco) on ice, blotted dry and weighed. Connective tissue wastrimmed away, and the ovaries were minced finely with a razor blade andwashed twice with EBSS 2×. Collagenase solution was prepared byvortexing 6.5 mg of collagenase (Sigma, Catologue # C 5138) in 5 ml ofEBSS. Minced tissue from 8 ovaries was added to 5 ml of collagenase inEBSS in a 50 ml Erlenmeyer flask and agitated gently by withdrawingseveral times into a Diamed pipette. The flask with minced tissue wasthen placed in a water bath at 37° C. for 20 minutes with gentle shaking(Position 45 on GFL incubator) under 95% air, 5% CO₂.

Following this incubation, the flask was placed on ice, and thesuspension of cells was transferred with a plastic transfer pipet onto aNitex filter fitted with Swiss Nitex Nylon Monofilament (75 m). Thefiltrate was collected into a 15 ml Falcon test tube. A second aliquot(2.5 ml) of collagenase solution (6.5 mg collagenase/5ml EBSS) was addedto the minced tissue remaining in the 50 ml Erlenmeyer flask, agitatedgently using a pipette, incubated for 10 minutes and filtered as above.The two filtrates were combined and centrifuged in a clinical centrifuge(˜200g) for 5 minutes at room temperature. All but ˜2ml of thesupernatant were removed with a pipet and discarded, and the sedimentedcells were resuspended in the remaining 2 ml of supernatant.

The cells were washed twice by adding 5 ml of MEM and centrifuging andresuspending as above. The washed cells were resuspended in 30 mls ofMEM containing 10 mm glutamine in a 50 ml Erlenmeyer flask and incubatedfor 1 hour without shaking at 37° C. under 95% air, 5% CO₂. The cellswere then sedimented by centrifugation as above and resuspended in MEMcontaining 10 mM glutamine.

The concentration of dispersed cells was determined using ahemocytometer, and viability was assessed using trypan blue dye.Aliquots of 2-5×10⁵ cells were placed in 12×75 mm test tubes andincubated without shaking for 2-5 hours at 37° C. under 95% air, 5% CO₂.The progress of apoptosis during this period was monitored by assessingthe degree of DNA laddering.

Visualization of Apoptosis in Rat Corpus Luteum by DNA Laddering

The degree of apoptosis was determined by DNA laddering. Genomic DNA wasisolated from dispersed corpus luteal cells or from excised corpusluteum tissue using the QIAamp DNA Blood Kit (Qiagen) according to themanufacturer's instructions. Corpus luteum tissue was excised before theinduction of apoptosis by treatment with PGF-2α, 1 and 24 hours afterinduction of apoptosis. The isolated DNA was end-labeled by incubating500 ng of DNA with 0.2 μCi [α-³²P]dCTP, i mM Tris, 0.5 mM EDTA, 3 unitsof Klenow enzyme, and 0.2 pM each of dATP, dGTP, and dTTP at roomtemperature for 30 minutes. Unincorporated nucleotides were removed bypassing the sample through a 1 ml Sepadex G-50 column according toSambrook et al. The samples were then resolved by Tris-acetate-EDTA(1.8%) gel electrophoresis. The gel was dried for 30 minutes at roomtemperature under vacuum and exposed to x-ray film at −80° C. for 24hours.

In one experiment, the degree of apoptosis in superovulated rat corpuslutea was examined either 0, 1, or 24 hours after injection with PGF-2α.In the 0 hour control, the ovaries were removed without PGF-2αinjection. Laddering of low molecular weight DNA fragments reflectingnuclease activity associated with apoptosis is not evident in controlcorpus luteum tissue excised before treatment with PGF-2α, but isdiscernible within 1 hour after induction of apoptosis and is pronouncedby 24 hours after induction of apoptosis, which is shown in FIG. 16. Inthis figure, the top panel is an autoradiograph of the Northern blotprobed with the ³²P-dCTP-labeled 3′-untranslated region of rat corpusluteum apoptosis-specific DHS cDNA. The lower panel is the ethidiumbromide stained gel of total RNA. Each lane contains 10 μg RNA. The dataindicate that there is down-regulation of eIF-5A transcript followingserum withdrawal.

In another experiment, the corresponding control animals were treatedwith saline instead of PGF-2α. Fifteen minutes after treatment withsaline or PGF-2α, corpora lutea were removed from the animals. GenomicDNA was isolated from the corpora lutea at 3 hours and 6 hours afterremoval of the tissue from the animals. DNA laddering and increased endlabeling of genomic DNA are evident 6 hours after removal of the tissuefrom the PGF-2α-treated animals, but not at 3 hours after removal of thetissue. See FIG. 17. DNA laddering reflecting apoptosis is also evidentwhen corpora lutea are excised 15 minutes after treatment with PGF-2αand maintained for 6 hours under in vitro conditions in EBSS (Gibco).Nuclease activity associated with apoptosis is also evident from moreextensive end labeling of genomic DNA.

In another experiment, superovulation was induced by subcutaneousinjection with 500 μg of PGF-2α. Control rats were treated with anequivalent volume of saline solution. Fifteen to thirty minutes later,the ovaries were removed and minced with collagenase. The dispersedcells from rats treated with PGF-2α were incubated in 10 mm glutamine+10mm spermidine for 1 hour and for a further 5 hours in 10 mm glutaminewithout spermidine (lane 2) or in 10 mm glutamine+10 mm spermidine for 1hour and for a further 5 hours in 10 mm glutamine+1 mm spermidine (lane3). Control cells from rats treated with saline were dispersed withcollagenase and incubated for 1 hour and a further 5 hours in glutamineonly (lane 1). Five hundred nanograms of DNA from each sample waslabeled with [α-³²P]-dCTP using Klenow enzyme, separated on a 1.8%agarose gel, and exposed to film for 24 hours. Results are shown in FIG.18.

In yet another experiment, superovulated rats were injectedsubcutaneously with 1 mg/100 g body weight of spermidine, delivered inthree equal doses of 0.333 mg/100 g body weight, 24, 12, and 2 hoursprior to a subcutaneous injection with 500 μg PGF-2α. Control rats weredivided into three sets: no injections, three injections of spermidinebut no PGF-2α; and three injections with an equivalent volume of salineprior to PGF-2α treatment. Ovaries were removed front the rats either 1hour and 35 minutes or 3 hours and 45 minutes after prostaglandintreatment and used for the isolation of DNA. Five hundred nanograms ofDNA from each sample was labeled with [α-³²P]-dCTP using Klenow enzyme,separated on a 1.8% agarose gel, and exposed to film for 24 hours: lane1, no injections (animals were sacrificed at the same time as for lanes3-5); lane 2, three injections with spermidine (animals were sacrificedat the same time as for lanes 3-5); lane 3, three injections with salinefollowed by injection with PGF-2α (animals were sacrificed 1 h and 35min after treatment with PGF-2α); lane 4, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 1h and 35 min after treatment with PGF-2α); lane 5, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 1h and 35 min after treatment with PGF-2α); lane 6, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 3h and 45 min after treatment with PGF-2α); lane 7, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 3h and 45 min after treatment with PGF-2α). Results are shown in FIG. 19.

RNA Isolation

Total RNA was isolated from corpus luteum tissue removed from rats atvarious times after PGF-2α induction of apoptosis. Briefly, the tissue(5 g) was ground in liquid nitrogen. The ground powder was mixed with 30ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH8.5, 0.8% β-mercaptoethanol). The mixture was filtered through fourlayers of Miracloth and centrifuged at 10,000 g at 4° C. for 30 minutes.The supernatant was then subjected to cesium chloride density gradientcentrifugation at 11,200 g for 20 hours. The pelleted RNA was rinsedwith 75% ethanol, resuspended in 600 ml DEPC-treated water and the RNAprecipitated at −70° C. with 1.5 ml 95% ethanol and 60 ml of 3M NaOAc.

Genomic DNA Isolation and Laddering

Genomic DNA was isolated from extracted corpus luteum tissue ordispersed corpus luteal cells using the QIAamp DNA Blood Kit (Qiagen)according to the manufacturer's instructions. The DNA was end-labeled byincubating 500 ng of DNA with 0.2 μCi [α-³²P]dCTP, 1 mM Tris, 0.5 mMEDTA, 3 units of Klenow enzyme, and 0.2 pM each of dATP, dGTP, and dTTP,at room temperature for 30 minutes. Unincorporated nucleotides wereremoved by passing the sample through a 1-ml Sephadex G-50 columnaccording to the method described by Maniatis et al. The samples werethen resolved by Tris-acetate-EDTA (2%) gel electrophoresis. The gel wasdried for 30 minutes at room temperature under vacuum and exposed tox-ray film at −80° C. for 24 hours.

Plasmid DNA Isolation, DNA Sequencing

The alkaline lysis method described by Sambrook et al., supra, was usedto isolate plasmid DNA. The full-length positive cDNA clone wassequenced using the dideoxy sequencing method. Sanger et al., Proc.Natl. Acad. Sci. USA, 74:5463-5467. The open reading frame was compiledand analyzed using BLAST search (GenBank, Bethesda, Md.) and sequencealignment was achieved using a BCM Search Launcher: Multiple SequenceAlignments Pattern-Induced Multiple Alignment Method (see F. Corpet,Nuc. Acids Res., 16:10881-10890, (1987). Sequences and sequencealignments are shown in FIGS. 5-11.

Northern Blot Hybridization of Rat Corpus Luteum RNA

Twenty milligrams of total RNA isolated from rat corpus luteum atvarious stages of apoptosis were separated on 1% denatured formaldehydeagarose gels and immobilized on nylon membranes. The full-length ratapoptosis-specific eIF-5A cDNA (SEQ ID NO:1) labeled with ³²P-dCTP usinga random primer kit (Boehringer) was used to probe the membranes 7×10⁷.Alternatively, full length rat apoptosis-specific DHS cDNA (SEQ ID NO:6)labeled with ³²P-dCTP using a random primer kit (Boehringer) was used toprobe the membranes (7×10⁷ cpm). The membranes were washed once with1×SSC, 0.1% SDS at room temperature and three times with 0.2×SSC, 0.1%SDS at 65° C. The membranes were dried and exposed to X-ray filmovernight at −70° C.

As can be seen, eIF-5A and DHS are both upregulated in apoptosing corpusluteum tissue. Expression of apoptosis-specific eIF-5A is significantlyenhanced after induction of apoptosis by treatment with PGF-2α—low attime zero, increased substantially within 1 hour of treatment, increasedstill more within 8 hours of treatment and increased slightly within 24hours of treatment (FIG. 14). Expression of DHS was low at time zero,increased substantially within 1 hour of treatment, increased still morewithin 8 hours of treatment and increased again slightly within 24 hoursof treatment (FIG. 15).

Generation of an Apoptosing Rat Corpus Luteum RT-PCR Product UsingPrimers Based on Yeast, Fungal and Human eIF-5A Sequences

A partial-length apoptosis-specific eIF-5A sequence (SEQ ID NO:11)corresponding to the 3′ end of the gene was generated from apoptosingrat corpus luteum RNA template by RT-PCR using a pair of oligonucleotideprimers designed from yeast, fungal and human eIF-5A sequences. Theupstream primer used to isolate the 3′end of the rat eIF-5A gene is a 20nucleotide degenerate primer: 5′ TCSAARACHGGNAAGCAYGG 3′ (SEQ ID NO:9),wherein S is selected from C and G; R is selected from A and G; H isselected from A, T, and C; Y is selected from C and T; and N is anynucleic acid. The downstream primer used to isolate the 3′end of the rateIF-5A gene contains 42 nucleotides: 5′ GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3′ (SEQ ID NO:10). A reverse transcriptasepolymerase chain reaction (RT-PCR) was carried out. Briefly, using 5 mgof the downstream primer, a first strand of cDNA was synthesized. Thefirst strand was then used as a template in a RT-PCR using both theupstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence a 900 bp fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using blunt end ligationand sequencted (SEQ ID NO:11). The cDNA sequence of the 3′ end is SEQ IDNO:11 and the amino acid sequence of the 3′ end is SEQ ID NO:12. SeeFIGS. 1-2.

A partial-length apoptosis-specific eIF-5A sequence (SEQ ID NO:15)corresponding to the 5′ end of the gene and overlapping with the 3′ endwas generated from apoptosing rat corpus luteum RNA template by RT-PCR.The 5′ primer is a 24-mer having the sequence, 5′CAGGTCTAGAGTTGGAATCGAAGC 3′ (SEQ ID NO: 13), that was designed fromhuman eIF-5A sequences. The 3′ primer is a 30-mer having the sequence,5′ ATATCTCGAGCCTT GATTGCAACAGCTGCC 3′ (SEQ ID NO:14) that was designedaccording to the 3′ end RT-PCR fragment. A reversetranscriptase-polymerase chain reaction (RT-PCR) was carried out.Briefly, using 5 mg of the downstream primer, a first strand of cDNA wassynthesized. The first strand was then used as a template in a RT-PCRusing both the upstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence a 500 bp fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LsJolla, Calif.) using XbaI and XhoIcloning sites present in the upstream and downstream primers,respectively, and sequenced (SEQ ID NO:15). The cDNA sequence of the 5′end is SEQ ID NO:15, and the amino acid sequence of the 5′ end is SEQ IDNO: 16. See FIG. 2.

The sequences of the 3′ and 5′ ends of the rat apoptosis-specific eIF-5A(SEQ ID NO:11 and SEQ ID NO:15, respectively) overlapped and gave riseto the full-length cDNA sequence (SEQ ID NO: 1). This full-lengthsequence was aligned and compared with sequences in the GeneBank database. See FIGS. 1-2. The cDNA clone encodes a 154 amino acid polypeptide(SEQ ID NO:2) having a calculated molecular mass of 16.8 KDa. Thenucleotide sequence, SEQ ID NO: 1, for the full length cDNA of the ratapoptosis-specific corpus luteum eIF-5A gene obtained by RT-PCR isdepicted in FIG. 3 and the corresponding derived amino acid sequence isSEQ ID NO:9. The derived full-length amino acid sequence of eIF-5A wasaligned with human and mouse eIF-5a sequences. See FIG. 7-9.

Generation of an Apoptosing Rat Corpus Luteum RT-PCR Product UsingPrimers Based on a Human DHS Sequence

A partial-length apoptosis-specific DHS sequence (SEQ ID NO:6)corresponding to the 3′ end of the gene was generated from apoptosingrat corpus luteum RNA template by RT-PCR using a pair of oligonucleotideprimers designed from a human DHS sequence. The 5′ primer is a 20-merhaving the sequence, 5′ GTCTGTGTATTATTGGGCCC 3′ (SEQ ID NO. 17); the 3′primer is a 42-mer having the sequence, 5′ GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3′ (SEQ ID NO: 18). A reverse transcriptasepolymerase chain reaction (RT-PCR) was carried out. Briefly, using 5 mgof the downstream primer, a first strand of cDNA was synthesized. Thefirst strand was then used as a template in a RT-PCR using both theupstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence a 606 bp fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using blunt end ligationand sequenced (SEQ ID NO:6). The nucleotide sequence (SEQ ID NO:6) forthe partial length cDNA of the rat apoptosis-specific corpus luteum DHSgene obtained by RT-PCR is depicted in FIG. 4 and the correspondingderived amino acid sequence is SEQ ID NO.7.

Isolation of Genomic DNA and Southern Analysis

Genomic DNA for southern blotting was isolated from excised rat ovaries.Approximately 100 mg of ovary tissue was divided into small pieces andplaced into a 15 ml tube. The tissue was washed twice with 1 ml of PBSby gently shaking the tissue suspension and then removing the PBS usinga pipette. The tissue was resuspended in 2.06 ml of DNA-buffer (0.2 MTris-HCl pH 8.0 and 0.1 mM EDTA) and 240 μl of 10% SDS and 100 μl ofproteinase K (Boehringer Manheim; 10 mg/ml) was added. The tissue wasplaced in a shaking water bath at 45° C. overnight. The following dayanother 100 μl of proteinase K (10 mg/ml) was added and the tissuesuspension was incubated in a waterbath at 45° C. for an additional 4hours. After the incubation the tissue suspension was extracted oncewith an equal volume of phenol:chloroform:iso-amyl alcohol (25:24:1) andonce with an equal volume of chloroform: iso-amyl alcohol (24:1).Following the extractions 1/10th volume of 3M sodium acetate (pH 5.2)and 2 volumes of ethanol were added. A glass pipette sealed and formedinto a hook using a Bunsen burner was used to pull the DNA threads outof solution and to transfer the DNA into a clean microcentrifuge tube.The DNA was washed once in 70% ethanol and air-dried for 10 minutes. TheDNA pellet was dissolved in 500 μl of 10 mM Tris-HCl (pH 8.0), 10 μl ofRNase A (10 mg/ml) was added, and the DNA was incubated for 1 hour at37° C. The DNA was extracted once with phenol:chloroform:iso-amylalcohol (25:24:1) and the DNA was precipitated by adding 1/10th volumeof 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. The DNA waspelleted by centrifugation for 10 minutes at 13,000×g at 4° C. The DNApellet was washed once in 70% ethanol and dissolved in 200 μl 10 mMTris-HCl (pH 8.0) by rotating the DNA at 4° C. overnight.

For Southern blot analysis, genomic DNA isolated from rat ovaries wasdigested with various restriction enzymes that either do not cut in theendogenous gene or cut only once. To achieve this, 10 μg genomic DNA, 20μl 10× reaction buffer and 100 U restriction enzyme were reacted forfive to six hours in a total reaction volume of 200 μl. Digested DNA wasloaded onto a 0.7% agarose gel and subjected to electrophoresis for 6hours at 40 volts or overnight at 15 volts. After electrophoresis, thegel was depurinated for 10 minutes in 0.2 N HCl followed by two15-minute washes in denaturing solution (0.5 M NaOH, 1.5 M NaCl) and two15 minute washes in neutralizing buffer (1.5 M NaCl, 0.5 M Tris-HCl pH7.4). The DNA was transferred to a nylon membrane, and the membrane wasprehybridized in hybridization solution (40% formamide, 6×SSC, 5×Denhart's, solution (1× Denhart's solution is 0.02% Ficoll, 0.02% PVP,and 0.02% BSA), 0.5% SDS, and 1.5 mg of denatured salmon sperm DNA). A700 bp PCR fragment of the 3′ UTR of rat eIF-5A cDNA (650 bp of 3′ UTRand 50 bp of coding) was labeled with [α-³²P]-dCTP by random priming andadded to the membrane at 1×106 cpm/ml.

Similarly, a 606 bp PCR fragment of the rat DHS cDNA (450 bp coding and156 bp 3′ UTR) was random prime labeled with [α-³²P]-dCTP and added at1×10 6 cpm/ml to a second identical membrane. The blots were hybridizedovernight at 42° C. and then washed twice with 2×SSC and 0.1% SDS at 42°C. and twice with 1×SSC and 0.1% SDS at 42° C. The blots were thenexposed to film for 3-10 days.

Rat corpus genomic DNA was cut with restriction enzymes as indicated onFIG. 20 and probed with ³²P-dCTP-labeled full-length eIF-5A cDNA.Hybridization under high stringency conditions revealed hybridization ofthe full-length cDNA probe to several restriction fragments for eachrestriction enzyme digested DNA sample, indicating the presence ofseveral isoforms of eIF-5A. Of particular note, when rat genomic DNA wasdigested with EcoRV, which has a restriction site within the openreading frame of apoptosis-specific eIF-5A, two restriction fragments ofthe apoptosis-specific isoform of eIF-5A were detectable in the Southernblot. The two fragments are indicated with double arrows in FIG. 20. Therestriction fragment corresponding to the apoptosis-specific isoform ofeIF-5A is indicated by a single arrow in the lanes labeled EcoR1 andBamH1, restriction enzymes for which there are no cut sites within theopen reading frame. These results suggest that the apoptosis-specificeIF-5A is a single copy gene in rat. As shown in FIGS. 5 through 13, theeIF-5A gene is highly conserved across species, and so it would beexpected that there is a significant amount of conservation betweenisoforms within any species.

FIG. 21 shows a Southern blot of rat genomic DNA probed with³²P-dCTP-labeled partial-length rat corpus luteum apoptosis-specific DHScDNA. The genomic DNA was cut with EcoRV, a restriction enzyme that doesnot cut the partial-length cDNA used as a probe. Two restrictionfragments are evident indicating that there are two copies of the geneor that the gene contains an intron with an EcoRV site.

Example 2

The present example demonstrates modulation of apoptosis withapoptosis-specific eIF-5A and DHS.

Culturing of COS-7 Cells and Isolation of RNA

COS-7, an African green monkey kidney fibroblast-like cell linetransformed with a mutant of SV40 that codes for wild-type T antigen,was used for all transfection-based experiments. COS-7 cells werecultured in Dulbecco's Modified Eagle's medium (DMEM) with 0.584 gramsper liter of L-glutamine, 4.5 g of glucose per liter, and 0.37% sodiumbicarbonate. The culture media was supplemented with 10% fetal bovineserum (FBS) and 100 units of penicillin/streptomycin. The cells weregrown at 37° C. in a humidified environment of 5% CO₂ and 95% air. Thecells were subcultured every 3 to 4 days by detaching the adherent cellswith a solution of 0.25% trypsin and 1 mM EDTA. The detached cells weredispensed at a split ratio of 1:10 in a new culture dish with freshmedia.

COS-7 cells to be used for isolation of RNA were grown in 150-mm tissueculture treated dishes (Coming). The cells were harvested by detachingthem with a solution of trypsin-EDTA. The detached cells were collectedin a centrifuge tube, and the cells were pelleted by centrifugation at3000 rpm for 5 minutes. The supernatant was removed, and the cell pelletwas flash-frozen in liquid nitrogen. RNA was isolated from the frozencells using the GenElute Mammalian Total RNA Miniprep kit (Sigma)according to the manufacturer's instructions.

Construction of Recombinant Plasmids and Transfection of COS-7 Cells

Recombinant plasmids carrying the full-length coding sequence of ratapoptosis eIF-5A in the sense orientation and the 3′ untranslated region(UTR) of rat apoptosis eIF-5A in the antisense orientation wereconstructed using the mammalian epitope tag expression vector, pHM6(Roche Molecular Biochemicals), which is illustrated in FIG. 21. Thevector contains the following: CMV promoter-human cytomegalovirusimmediate-early promoter/enhancer; HA-nonapeptide epitope tag frominfluenza hemagglutinin; BGH pA-Bovine growth hormone polyadenylationsignal; f1 ori-f1 origin; SV40 ori-SV40 early promoter and origin;Neomycin-Neomycin resistance (G418) gene; SV40 pA-SV40 polyadenylationsignal; Col E1-ColE1 origin; Ampicillin-Ampicillin resistance gene. Thefull-length coding sequence of rat apoptosis eIF-5A and the 3′ UTR ofrat apoptosis eIF-5A were amplified by PCR from the original rat eIF-5ART-PCR fragment in pBluescript (SEQ ID NO:1). To amplify the full-lengtheIF-5A the primers used were as follows: Forward 5′GCCAAGCTTAATGGCAGATGATTT GG 3′ (Hind3)(SEQ ID NO:22) and Reverse 5′CTGAATTCCAGT TATTTTGCCATGG 3′ (EcoR1)(SEQ ID NO:23). To amplify the 3′UTR rat eIF-5A the primers used were as follows: forward 5′AATGAATTCCGCCATGACAGAGGAGGC 3′ (EcoR1)(SEQ ID NO:24) and reverse 5′GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3′ (Hind3)(SEQ ID NO: 10).

The full-length rat eIF-5A PCR product isolated after agarose gelelectrophoresis was 430 bp in length while the 3′ UTR rat eIF-5A PCRproduct was 697 bp in length. Both PCR products were subcloned into theHind 3 and EcoR1 sites of pHM6 to create pHM6-full-length eIF-5A andpHM6-antisense 3′UTReIF-5A. The full-length rat eIF-5A PCR product wassubcloned in frame with the nonapeptide epitope tag from influenzahemagglutinin (HA) present upstream of the multiple cloning site toallow for detection of the recombinant protein using ananti-[HA]-peroxidase antibody. Expression is driven by the humancytomegalovirus immediate-early promoter/enhancer to ensure high levelexpression in mammalian cell lines. The plasmid also features aneomycin-resistance (G418) gene, which allows for selection of stabletransfectants, and a SV40 early promoter and origin, which allowsepisomal replication in cells expressing SV40 large T antigen, such asCOS-7.

COS-7 cells to be used in transfection experiments were cultured ineither 24 well cell culture plates (Corning) for cells to be used forprotein extraction, or 4 chamber culture slides (Falcon) for cells to beused for staining. The cells were grown in DMEM media supplemented with10% FBS, but lacking penicillin/streptomycin, to 50 to 70% confluency.Transfection medium sufficient for one well of a 24-well plate orculture slide was prepared by diluting 0.32 μg of plasmid DNA in 42.5 μlof serum-free DMEM and incubating the mixture at room temperature for 15minutes. 1.6 μl of the transfection reagent, LipofectAMINE (Gibco, BRL),was diluted in 42.5 μl of serum-free DMEM and incubated for 5 minutes atroom temperature. After 5 minutes the LipofectAMINE mixture was added tothe DNA mixture and incubated together at room temperature for 30 to 60minutes. The cells to be transfected were washed once with serum-freeDMEM before overlaying the transfection medium and the cells were placedback in the growth chamber for 4 hours.

After the incubation, 0.17 ml of DMEM+20% FBS was added to the cells.The cells were the cultured for a further 40 hours before either beinginduced to undergo apoptosis prior to staining or harvested for Westernblot analysis. As a control, mock transfections were also performed inwhich the plasmid DNA was omitted from the transfection medium.

Protein Extraction and Western Blotting

Protein was isolated for Western blotting from transfected cells bywashing the cells twice in PBS (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/LNa₂HPO₄, and 0.24 g/L KH₂PO₄) and then adding 150 μl of hot SDSgel-loading buffer (50 mM Tris-HCl pH 6.8, 100 mM dithiothreitol, 2%SDS, 0.1% bromophenol blue, and 10% glycerol). The cell lysate wascollected in a microcentrifuge tube, heated at 95° C. for 10 minutes,and then centrifuged at 13,000×g for 10 minutes. The supernatant wastransferred to a fresh microcentrifuge tube and stored at −20° C. untilready for use.

For Western blotting, 2.5 or 5 μg of total protein was separated on a12% SDS-polyacrylamide gel. The separated proteins were transferred to apolyvinylidene difluoride membrane. The membrane was then incubated forone hour in blocking solution (5% skim milk powder, 0.02% sodium azidein PBS) and washed three times for 15 minutes in PBS-T (PBS+0.05%Tween-20). The membrane was stored overnight in PBS-T at 4° C. Afterbeing warmed to room temperature the next day, the membrane was blockedfor 30 seconds in 1 μg/ml polyvinyl alcohol. The membrane was rinsed 5times in deionized water and then blocked for 30 minutes in a solutionof 5% milk in PBS. The primary antibody was preincubated for 30 minutesin a solution of 5% milk in PBS prior to incubation with the membrane.

Several primary antibodies were used. An anti-[HA]-peroxidase antibody(Roche Molecular Biochemicals) was used at a dilution of 1:5000 todetect expression of the recombinant proteins. Since this antibody isconjugated to peroxidase, no secondary antibody was necessary, and theblot was washed and developed by chemiluminescence. The other primaryantibodies that were used are monoclonal antibodies from Oncogene thatrecognize p53 (Ab-6), Bcl-2 (Ab-1), and c-Myc (Ab-2). The monoclonalantibody to p53 was used at a dilution of 0.1 μg/ml, and the monoclonalantibodies to Bcl-2 and c-Myc were both used at a dilution of 0.83μg/ml. After incubation with primary antibody for 60 to 90 minutes, themembrane was washed 3 times for 15 minutes in PBS-T. Secondary antibodywas then diluted in 1% milk in PBS and incubated with the membrane for60 to 90 minutes. When p53 (Ab-6) was used as the primary antibody, thesecondary antibody used was a goat anti-mouse IgG conjugated to alkalinephosphatase (Rockland) at a dilution of 1:1000. When Bcl-2 (Ab-1) andc-Myc (Ab-2) were used as the primary antibody, a rabbit anti-mouse IgGconjugated to peroxidase (Sigma) was used at a dilution of 1:5000. Afterincubation with the secondary antibody, the membrane was washed 3 timesin PBS-T.

Two detection methods were used to develop the blots, a colorimetricmethod and a chemiluminescent method. The colorimetric method was usedonly when p53 (Ab-6) was used as the primary antibody in conjunctionwith the alkaline phosphatase-conjugated secondary antibody. Boundantibody was visualized by incubating the blot in the dark in a solutionof 0.33 mg/mL nitro blue tetrazolium, 0.165 mg/mL5-bromo-4-chloro-3-indolyl phosphate, 100 mM NaCl, 5 mM MgCl₂, and 100mM Tris-HCl (pH 9.5). The color reaction was stopped by incubating theblot in 2 mM EDTA in PBS. A chemiluminescent detection method was usedfor all other primary antibodies, including anti-[HA]-peroxidase, Bcl-2(Ab-1), and c-Myc (Ab-2). The ECL Plus Western blotting detection kit(Amersham Pharmacia Biotech) was used to detect peroxidase-conjugatedbound antibodies. In brief, the membrane was lightly blotted dry andthen incubated in the dark with a 40:1 mix of reagent A and reagent Bfor 5 minutes. The membrane was blotted dry, placed between sheets ofacetate, and exposed to X-ray film for time periods varying from 10seconds to 10 minutes.

Induction of Apoptosis in COS 7 Cells

Two methods were used to induce apoptosis in transfected COS-7 cells,serum deprivation and treatment with Actinomycin D, streptomyces sp(Calbiochem). For both treatments, the medium was removed 40 hourspost-transfection. For serum starvation experiments, the media wasreplaced with serum- and antibiotic-free DMEM. Cells grown inantibiotic-free DMEM supplemented with 10% FBS were used as a control.For Actinomycin D induction of apoptosis, the media was replaced withantibiotic-free DMEM supplemented with 10% FBS and 1 μg/ml Actinomycin Ddissolved in methanol. Control cells were grown in antibiotic-free DMEMsupplemented with 10% FBS and an equivalent volume of methanol. For bothmethods, the percentage of apoptotic cells was determined 48 hours laterby staining with either Hoescht or Annexin V-Cy3. Induction of apoptosiswas also confirmed by Northern blot analyses, as shown in FIG. 22.

Hoescht Staining

The nuclear stain, Hoescht, was used to label the nuclei of transfectedCOS-7 cells in order to identify apoptotic cells based on morphologicalfeatures such as nuclear fragmentation and condensation. A fixative,consisting of a 3:1 mixture of absolute methanol and glacial aceticacid, was prepared immediately before use. An equal volume of fixativewas added to the media of COS-7 cells growing on a culture slide andincubated for 2 minutes. The media/fixative mixture was removed from thecells and discarded, and 1 ml of fixative was added to the cells. After5 minutes the fixative was discarded, and 1 ml of fresh fixative wasadded to the cells and incubated for 5 minutes. The fixative wasdiscarded, and the cells were air-dried for 4 minutes before adding 1 mlof Hoescht stain (0.5 μg/ml Hoescht 33258 in PBS). After a 10-minuteincubation in the dark, the staining solution was discarded and theslide was washed 3 times for 1 minute with deionized water. Afterwashing, 1 ml of McIlvaine's buffer (0.021 M citric acid, 0.058 MNa₂HPO₄.7H₂O; pH 5.6) was added to the cells, and they were incubated inthe dark for 20 minutes. The buffer was discarded, the cells wereair-dried for 5 minutes in the dark and the chambers separating thewells of the culture slide were removed. A few drops of Vectashieldmounting media for fluorescence (Vector Laboratories) was added to theslide and overlaid with a coverslip. The stained cells were viewed undera fluorescence microscope using a UV filter. Cells with brightly stainedor fragmented nuclei were scored as apoptotic.

Annexin V-Cy3 Staining

An Annexin V-Cy3 apoptosis detection kit (Sigma) was used tofluorescently label externalized phosphatidylserine on apoptotic cells.The kit was used according to the manufacturer's protocol with thefollowing modifications. In brief, transfected COS-7 cells growing onfour chamber culture slides were washed twice with PBS and three timeswith 1× Binding Buffer. 150 μl of staining solution (1 μg/ml AnnCy3 in1× Binding Buffer) was added, and the cells were incubated in the darkfor 10 minutes. The staining solution was then removed, and the cellswere washed 5 times with 1× Binding Buffer. The chamber walls wereremoved from the culture slide, and several drops of 1× Binding Bufferwere placed on the cells and overlaid with a coverslip. The stainedcells were analyzed by fluorescence microscopy using a green filter tovisualize the red fluorescence of positively stained (apoptotic) cells.The total cell population was determined by counting the cell numberunder visible light.

Example 3

The present example demonstrates modulation of apoptosis withapoptosis-specific eIF-5A and DHS.

Using the general procedures and methods described in the previousexamples, FIG. 23 is a flow chart illustrating the procedure fortransient transfection of COS-7 cells, in which cells in serum-freemedium were incubated in plasmid DNA in lipofectAMINE for 4 hours, serumwas added, and the cells were incubated for a further 40 hours. Thecells were then either incubated in regular medium containing serum fora further 48 hours before analysis (i.e. no further treatment), deprivedof serum for 48 hours to induce apoptosis before analysis, or treatedwith actinomycin D for 48 hours to induce apoptosis before analysis.

FIG. 22 is a Western blot illustrating transient expression of foreignproteins in COS-7 cells following transfection with pHM6. Protein wasisolated from COS-7 cells 48 hours after either mock transfection, ortransfection with pHM6-LacZ, pHM6-Antisense 3′ rF5A (pHM6-Antisense 3′UTR rat apoptosis eIF-5A), or pHM6-Sense rF5A (pHM6-Full length ratapoptosis eIF-5A). Five μg of protein from each sample was fractionatedby SDS-PAGE, transferred to a PVDF membrane, and Western blotted withanti-[HA]-peroxidase. The bound antibody was detected bychemiluminescence and exposed to x-ray film for 30 seconds. Expressionof LacZ (lane 2) and of sense rat apoptosis eIF-5A (lane 4) is clearlyvisible.

As described above, COS-7 cells were either mock transfected ortransfected with pHM6-Sense rF5A (pHM6-Full length rat eIF-5A). Fortyhours after transfection, the cells were induced to undergo apoptosis bywithdrawal of serum for 48 hours. The caspase proteolytic activity inthe transfected cell extract was measured using a fluorometrichomogenous caspase assay kit (Roche Diagnostics). DNA fragmentation wasalso measured using the FragEL DNA Fragmentation Apoptosis Detection kit(Oncogene) which labels the exposed 3′-OH ends of DNA fragments withfluorescein-labeled deoxynucleotides.

Additional COS-7 cells were either mock transfected or transfected withpHM6-Sense rF5A (pHM6-Full length rat eIF-5A). Forty hours aftertransfection, the cells were either grown for an additional 48 hours inregular medium containing serum (no further treatment), induced toundergo apoptosis by withdrawal of serum for 48 hours or induced toundergo apoptosis by treatment with 0.5 μg/ml of Actinomycin D for 48hours. The cells were either stained with Hoescht 33258, which depictsnuclear fragmentation accompanying apoptosis, or stained with AnnexinV-Cy3, which depicts phosphatidylserine exposure accompanying apoptosis.Stained cells were also viewed by fluorescence microscopy using a greenfilter and counted to determine the percentage of cells undergoingapoptosis. The total cell population was counted under visible light.

FIG. 25 illustrates enhanced apoptosis as reflected by increased caspaseactivity when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a60% increase in caspase activity.

FIG. 26 illustrates enhanced apoptosis as reflected by increased DNAfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a273% increase in DNA fragmentation. FIG. 27 illustrates detection ofapoptosis as reflected by increased nuclear fragmentation when COS-7cells were transiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. There is a greaterincidence of fragmented nuclei in cells expressing rat apoptosis-inducedeIF-5A. FIG. 28 illustrates enhanced apoptosis as reflected by increasednuclear fragmentation when COS-7 cells were transiently transfected withpHM6 containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a27% and 63% increase in nuclear fragmentation over control in non-serumstarved and serum starved samples, respectively.

FIG. 29 illustrates detection of apoptosis as reflected byphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-inducedeIF-5A in the sense orientation. FIG. 30 illustrates enhanced apoptosisas reflected by increased phosphatidylserine exposure when COS-7 cellswere transiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. Expression of ratapoptosis-induced eIF-5A resulted in a 140% and 198% increase inphosphatidylserine exposure over control, in non-serum starved and serumstarved samples, respectively.

FIG. 31 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-induced eIF-5A in the senseorientation. Expression of rat apoptosis-induced eIF-5A resulted in a115% and 62% increase in nuclear fragmentation over control in untreatedand treated samples, respectively. FIG. 32 illustrates a comparison ofenhanced apoptosis under conditions in which COS-7 cells transientlytransfected with pHM6 containing full-length rat apoptosis-inducedeIF-5A in the sense orientation were either given no further treatmentor treatment to induce apoptosis.

Example 4

The present example demonstrates modulation of apoptotic activityfollowing administration of apoptosis-specific eIF-5A and DHS.

Moreover, COS-7 cells were either mock transfected, transfected withpHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-Full length rateIF-5A) and incubated for 40 hours. Five μg samples of protein extractfrom each sample were fractionated by SDS-PAGE, transferred to a PVDFmembrane, and Western blotted with a monoclonal antibody that recognizesBcl-2. Rabbit anti-mouse IgG conjugated to peroxidase was used as asecondary antibody, and bound antibody was detected by chemiluminescenceand exposure to x-ray film. Results are shown in FIG. 32. Less Bcl-2 isdetectable in cells transfected with pHM6-Sense rF5A than in thosetransfected with pHM6-LacZ; therefore, Bcl-2 is down-regulated.

Additional COS-7 cells were either mock transfected, transfected withpHM6-antisense 3′ rF5A (pHM6-antisense 3′ UTR of rat apoptosis-specificeIF-5A) or transfected with pHM6-Sense rF5A (pHM6-Full length ratapoptosis-specific eIF-5A). Forty hours after transfection, the cellswere induced to undergo apoptosis by withdrawal of serum for 48 hours.Five μg samples of protein extract from each sample were fractionated bySDS-PAGE, transferred to a PVDF membrane, and Western blotted with amonoclonal antibody that recognizes Bcl-2. Rabbit anti-mouse IgGconjugated to peroxidase was used as a secondary antibody, and boundantibody was detected by chemiluminescence and exposure to x-ray film.

Also additionally, COS-7 cells were either mock transfected, transfectedwith pHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-Full length rateIF-5A) and incubated for 40 hours. Five μg samples of protein extractfrom each sample were fractionated by SDS-PAGE, transferred to a PVDFmembrane, and Western blotted with a monoclonal antibody that recognizesp53. Goat anti-mouse IgG conjugated to alkaline phosphatase was used asa secondary antibody, and bound antibody was detected acolorimetrically.

Finally, COS-7 cells were either mock transfected, transfected withpHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-Full length rateIF-5A) and incubated for 40 hours. Five μg samples of protein extractfrom each sample were fractionated by SDS-PAGE, transferred to a PVDFmembrane, and probed with a monoclonal antibody that recognizes p53.Corresponding protein blots were probed with with anti-[HA]-peroxidaseto determine the level of rat apoptosis-specific eIF-5A expression. Goatanti-mouse IgG conjugated to alkaline phosphatase was used as asecondary antibody, and bound antibody was detected bychemiluminescence.

FIG. 33 illustrates downregulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. Less Bcl-2 is detectable incells transfected with pHM6-Sense rF5A than in those transfected withpHM6-LacZ.

FIG. 34 illustrates upregulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the antisense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. More Bcl-2 is detectable incells transfected with pHM6-antisense 3′ rF5A than in those mocktransfected or transfected with pHM6-Sense rF5A.

FIG. 35 illustrates upregulation of c-Myc when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. More c-Myc is detectable incells transfected with pHM6-Sense rF5A than in those transfected withpHM6-LacZ or the mock control.

FIG. 36 illustrates upregulation of p53 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-induced eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. More p53 is detectable incells transfected with pHM6-Sense rF5A than in those transfected withpHM6-LacZ or the mock control.

FIG. 37 illustrates the dependence of p53 upregulation upon theexpression of pHM6-full length rat apoptosis-induced eIF-5A in COS-7cells. In the Western blot probed with anti-[HA]-peroxidase, the upperpanel illustrates the Coomassie-blue-stained protein blot and the lowerpanel illustrates the corresponding Western blot. More ratapoptosis-induced eIF-5A is detectable in the first transfection than inthe second transfection. In the Western blot probed with anti-p53, theupper panel in A illustrates a corresponding Coomassie-blue-stainedprotein blot and the lower panel illustrates the Western blot with p53.For the first transfection, more p53 is detectable in cells transfectedwith pHM6-Sense rF5A than in those transfected with pHM6-LacZ or themock control. For the second transfection in which there was lessexpression of rat apoptosis-induced eIF-5A, there was no detectabledifference in levels of p53 between cells transfected with pHM6-SenserF5A, pHM6-LacZ or the mock control.

1. An expression vector comprising a polynucleotide encoding anapoptosis-specific eIF-5A and a promoter sequence operably linked to thepoynucleotide wherein the apoptosis-specific eIF-5A comprises SEQ IDNO:1.
 2. The expression vector of claim 1 wherein the promoter comprisesthe human cytomegalovirus immediate-early promoter/enhancer.